STARS - a muscle-specific actin-binding protein

ABSTRACT

The present invention relates to a new polypeptide and the gene coding therefore, gene being an evolutionarily conserved actin-binding protein, called STARS (striated muscle activator of Rho signalin), that is expressed specifically in cardiac and skeletal muscle cells and is upregulated in response to calcineurin signaling during cardiac hypertrophy. STARS is localized to the thin filament of the sarcomere and to actin stress fibers where it promotes actin bundling. STARS stimulates the transcriptional activity of serum response factor (SRF) through a mechanism that requires actin bundling and Rho kinase activation. STARS provides a mechanism for selectively enhancing the transcriptional activity of SRF in muscle cells and for linking changes in actin dynamics to gene transcription. Also disclosed are methods of using the gene and protein in drug screening and therapy, including, for example, use of the gene in gene therapy to treat cardiovascular disease.

[0001] The government may own rights in the present invention pursuantto grant number RO1HL61544 from the National Institute of Health. Thepresent application claims benefit of priority to U.S. ProvisionalSerial No. 60/404,706, filed Aug. 20, 2002, the entire contents of whichare hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields ofdevelopmental biology and molecular biology. More particularly, itconcerns an actin binding protein expressed specifically in striatedmuscle tissue.

[0004] 2. Description of Related Art

[0005] The actin cytoskeleton influences diverse cellular processes,including motility, mitosis, contractility, cytokinesis, endocytosis andsecretion (Burridge and Chrzanowska-Wodnicka, 1996; Schmidt and Hall,1998; Pantaloni et al., 2001). Actin also participates in numeroustransmembrane signaling systems by forming complexes with cell adhesionmolecules and receptors (Juliano and Haskill, 1993; Calderwood et al.,2000). In addition, actin has been implicated in the control of genetranscription through its direct association with chromatin remodelingcomplexes (Rando et al., 2000) and through indirect mechanisms mediatedby changes in cytoskeletal actin dynamics (Sotiropoulos et al., 1999).

[0006] Actin exists in monomeric (G-actin) and polymerized (F-actin)forms. The distribution of actin between these two forms is tightlyregulated and is influenced by numerous actin-binding proteins whichcontrol actin dynamics by severing (i.e. ADF/cofilin), cross-linking(i.e. actinin, tropomyosin), and capping (i.e. tropomodulin at the pointends and capZ at the Z-line) actin (Cooper and Schafer, 2000). Membersof the Rho GTPase family regulate cytoskeletal organization bystimulating actin polymerization and stress fiber formation whenactivated by extracellular signaling (Ridley and Hall, 1992). A numberof Rho effector molecules, including Rho kinase/ROCK, mDia andphosphatidylinositol phosphate 5-kinase also participate in cytoskeletalorganization (Kaibuchi et al., 1999; Maekawa et al., 1999; Narumiya etal., 1997; Yamamoto et al., 2001; Van Aelst and D'Souza-Schorey, 1997).

[0007] Recent studies showed that RhoA signaling stimulates thetranscriptional activity of serum response factor (SRF) through amechanism mediated by changes in actin dynamics (Sotiropoulos et al.,1999; Mack et al., 2001). SRF is a MADS-box transcription factor thatregulates serum-inducible and muscle-specific gene expression by bindingto a consensus sequence known a CArG box, CC(A/T)6GG (Treisman, 1995a;Treisman, 1995b). The spectrum of genes regulated by SRF is dictated byits association with serum-regulated and muscle-restricted cofactors(Treisman, 1994). In light of the unique and elaborate cytoskeletalorganization of striated muscle cells, and the sensitivity of SRF toactin dynamics, it is tempting to speculate that cytoskeletal signals instriated muscle cells might regulate SRF activity in a muscle-specificmanner.

SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides an isolated STARSpolypeptide, in particular a STARS polypeptide comprising an amino acidsequence of SEQ ID NO:2, 4, 6, 8 or 10. By way of illustration, thepolynucleotide may have the nucleic acid sequence of SEQ ID NO:1, 3, 5,7 or 9, or a complement thereof. The polynucleotide may further comprisea promoter operable in eukaryotic cells, for example, a promoterheterologous to the natural sequence of SEQ ID NO:1, 3, 5, 7 or 9.Exemplary promoters include hsp68, SV40, CMV, MKC, GAL4_(UAS), HSV, andβ-actin. The promoter may be a tissue specific promoter, such as acardiac specific promoter.

[0009] In another embodiment, there is provided a nucleic acid of 15 toabout 2000 base pairs comprising from about 15, 20, 25, 30, 40, 50, 100,150, 250, 500, 1000, 2000 or more contiguous base pairs of SEQ ID NO:1,3, 5, 7 or 9, or the complement thereof. Also provided is a peptidecomprising 10, 15, 20, 25, 30, 50 or more contiguous amino acids of SEQID NO:2, 4, 6, 8 or 10.

[0010] In yet another embodiment, there is provided an expressionconstruct comprising a polynucleotide encoding a STARS polypeptideoperably linked to a regulatory sequence, for example, a STARSpolypeptide having the sequence of SEQ ID NO:2, 4, 6, 8 or 10. Inparticular embodiments, the polynucleotide within the expressionconstruct is under the control of a tissue specific promoter operable ineukaryotic cells where the promoter may be muscle specific. Exemplarytissue specific promoters include myosin light chain-2 promoter, α actinpromoter, troponin 1 promoter, Na⁺/Ca²⁺ exchanger promoter, dystrophinpromoter, creatine kinase promoter, α7 integrin promoter, brainnatriuretic peptide promoter, αB-crystallin/small heat shock proteinpromoter, α myosin heavy chain promoter and atrial natriuretic factorpromoter. The promoter may be an inducible promoter.

[0011] The expression construct may be comprised within a viral vector,for example, a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, a herpesviralvector, a polyoma viral construct or a Sindbis viral vector. Theexpression construct may further comprise various regulatory sequences,such as, for example, a polyadenylation signal or the like. Theexpression construct may comprise a one or more additionalpolynucleotides encoding one or more additional polypeptides, under thecontrol of the same or a different promoter.

[0012] In still another embodiment, there is provided a method ofscreening for modulators of STARS expression comprising (a) providing acell in which a STARS promoter directs the expression of a polypeptide;(b) contacting said cell with a candidate modulator; and (c) measuringthe effect of said candidate modulator on said polypeptide, wherein adifference in expression of said polypeptide, as compared to anuntreated cell, indicates that said candidate modulator is a modulatorof STARS expression. Measuring may comprise Northern analysis, PCR,RT-PCR, or immunologic detection of STARS (including ELISA andimmunohistochemistry). The cell may be located in an animal. The celltype may be a myocyte, or more specifically, a cardiomyocyte. Themodulator may increase or decrease expression. The polypeptide may beSTARS or a screenable marker polypeptide.

[0013] In still yet another embodiment, there is provided a method ofscreening for modulators of STARS actin binding activity comprising (a)providing an active STARS preparation; (b) contacting said STARSpreparation with a candidate modulator; and (c) measuring the actinbinding activity of said STARS preparation, wherein a difference inactin binding activity of said STARS preparation, as compared to anuntreated STARS preparation, indicates that said candidate modulator isa modulator of STARS actin binding activity. The screening may beperformed in a cell free assay or in a cell. The binding may bedetermined by chromatographic separation or electrophoretic separation.

[0014] Further embodiments include a method of screening for aninhibitor of STARS-induced transcription comprising (a) providing a cellthat expresses STARS and contains a STARS-regulated promoter linked toan indicator gene; (b) contacting said cell with a candidate modulator;and (c) measuring the effect of said candidate modulator on expressionof said indicator gene, wherein a difference in expression of saidpolypeptide, as compared to an untreated cell, indicates that saidcandidate modulator is a modulator of STARS-induced transactivation. Thecell may be a myocyte, for example, a cardiomyocyte. The promoter may bean SM22 promoter. The indicator gene may encode luciferase,β-galactosidase, CAT, or green fluorescent protein.

[0015] Also provided is a method of producing a STARS polypeptide in acell comprising (a) transforming a cell with an expression cassettecomprising a nucleic acid encoding STARS under the control of a promoteractive in said cell; (b) culturing said cell under conditions suitablefor expression of STARS. The cell may be, for example, a cardiomyocyteor a fibroblast, such as a cardiac fibroblast. The cell may be locatedin an animal. The transforming step may comprise infection with a viralvector. The transforming step may also comprise contacting the cell witha liposome comprising the expression cassette, electroporation, calciumphosphate precipitation, or protoplast fusion. The cell may be aprokaryotic or eukaryotic cell. The method may further comprise the stepof purifying said STARS polypeptide away from other cellular components.

[0016] In other embodiments, there are provided a non-human transgenicanimal comprising a selectable or screenable marker protein under thecontrol of a STARS promoter; a non-human transgenic animal comprising aSTARS encoding nucleic acid under the control of an inducible promoter;a non-human transgenic animal comprising a STARS encoding nucleic acidunder the control of a constitutive promoter; and a non-human transgenicanimal lacking at least one STARS allele, or both.

[0017] In other embodiments, there are provided a method of inhibitingSTARS activity comprising contacting a cell expressing STARS with acompound that inhibits STARS activity. The compound may be a nucleicacid antisense to a STARS regulatory or coding region, a ribozyme thatselectively cleaves a STARS transcript, a small molecule inhibitor, or asingle chain antibody that binds immunologically to STARS.

[0018] In yet other embodiments, methods of treating cardiac hypertrophyand dilated cardiomyopathy comprising decreasing STARS activity in heartcells of a subject are provided. In one aspect, STARS activity isdecreased by delivering an expression vector comprising a polynucleotideencoding an antisense STARS construct, a STARS ribozyme or an anti-STARSsingle-chain antibody to said subject. The expression vector may be anon-viral or a viral vector. The viral vector may be an adenoviralconstruct, a retroviral construct, an adeno-associated viral construct,a herpesviral construct, a vaccinia viral construct, a polyoma viralconstruct or a Sindbis viral vector. The viral vector may be areplication-defective adenovirus.

[0019] By way of illustration, the step of delivering the expressionconstruct may comprise introducing a viral vector comprising the nucleicacid into the heart of the mammal by direct injection into the hearttissue. The step of delivering the expression construct may alsocomprise introducing the expression construct into the lumen of at leastone vessel that supplies blood to the heart. Delivering the expressionconstruct may further comprise administering a second anti-hypertophicdrug, or decreasing STARS activity in hearts cells of a subject.

[0020] In additional embodiments, there are provided:

[0021] a method of producing a modulator of STARS expression comprising:(a) providing a cell in which a STARS promoter directs the expression ofa polypeptide; (b) contacting said cell with a candidate modulator; (c)measuring the effect of said candidate modulator on said polypeptide,wherein a difference in expression of said polypeptide, as compared toan untreated cell, indicates that said candidate modulator is amodulator of STARS expression; and (d) producing said modulator;

[0022] a method of producing a modulator of STARS actin binding activitycomprising: (a) providing an active STARS preparation; (b) contactingsaid STARS preparation with a candidate modulator; (c) measuring theactin binding activity of said STARS preparation, wherein a differencein actin binding activity of said STARS preparation, as compared to anuntreated STARS preparation, indicates that said candidate modulator isa modulator of STARS acting binding activity; and (d) producing saidmodulator;

[0023] a modulator of STARS expression identified according to themethod comprising: (a) providing a cell in which a STARS promoterdirects the expression of a polypeptide; (b) contacting said cell with acandidate modulator; and (c) measuring the effect of said candidatemodulator on said polypeptide, wherein a difference in expression ofsaid polypeptide, as compared to an untreated cell, indicates that saidcandidate modulator is a modulator of STARS expression;

[0024] a modulator of STARS actin binding activity identified accordingto the method comprising: (a) providing a STARS preparation; (b)contacting said STARS preparation with a candidate modulator; and (c)measuring the actin binding activity of said STARS preparation, whereina difference in actin binding activity of said STARS preparation, ascompared to an untreated STARS preparation, indicates that saidcandidate modulator is a modulator of STARS actin binding activity;

[0025] There also are provided an antibody that binds immunologically toSTARS, a polyclonal antibody preparation of antibodies that bindimmunologically to STARS, and a hybridoma cell that produces amonoclonal antibody that binds immunologically to STARS.

[0026] In other embodiments, there are provided a method of treatingmyocardial infarct comprising decreasing STARS activity in heart cellsof a subject; a method of preventing cardiac hypertrophy and dilatedcardiomyopathy comprising decreasing STARS activity in heart cells of asubject; a method of inhibiting progression of cardiac hypertrophycomprising decreasing STARS activity in heart cells of a subject; amethod of treating heart failure comprising decreasing STARS activity inheart cells of a subject; a method of inhibiting progression of heartfailure comprising decreasing STARS activity in heart cells of asubject; a method of increasing exercise tolerance in a subject withheart failure or cardiac hypertrophy comprising decreasing STARSactivity in heart cells of a subject; a method of reducinghospitalization in a subject with heart failure or cardiac hypertrophycomprising decreasing STARS activity in heart cells of a subject; amethod of improving quality of life in a subject with heart failure orcardiac hypertrophy comprising decreasing STARS activity in heart cellsof a subject; a method of decreasing morbidity in a subject with heartfailure or cardiac hypertrophy comprising decreasing STARS activity inheart cells of a subject; and a method of decreasing mortality in asubject with heart failure or cardiac hypertrophy comprising decreasingSTARS in heart cells of a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0028]FIG. 1—Deduced amino acid sequence and homology of STARS to otherpredicted proteins. Deduced amino acid sequence of mouse STARS(Accession numbers AF504061 and AAM28877) and homology with sequencesfrom other species, including human (AF503617 and AAM27268).

[0029]FIG. 2—Expression of STARS in cardiac and skeletal muscle. FIG.2A: In situ hybridization shows the specific expression of STARStranscripts in the heart tube of E8.75 mouse embryo h, head; ht, heart.FIG. 2B: Northern blot analysis of STARS transcripts in adult mouse andhuman tissues detected with Clontech multi tissue blots. FIG. 2C:Northern blot analysis of STARS and GAPDH transcripts duringdifferentiation of the C2 muscle cell line. At day 0, myoblasts weremaintained in growth medium and were then transferred to differentiationmedium for the indicated number of days. FIG. 2D: Northern analysis ofSTARS and GAPDH transcripts in hearts from wild-type (WT) andα-MHC-calcineurin transgenic (Tg) mice at 3 and 6 months of age. Twentyμg total RNA was applied to each lane in FIG. 2C and FIG. 2D. Positionsof 18S and 28S RNA are indicated.

[0030]FIG. 3—Identification of STARS as a sarcomeric protein. FIG. 3A:Western blot analysis using anti-STARS antibody detected endogenousSTARS protein in adult mouse heart lysate and in COS cells transfectedwith STARS expression vector. FIG. 3B: Localization of STARS to theactin thin-filament and stress fibers in cardiomyocytes. Rat neonatalcardiomyocytes were infected with GFP adenovirus (a, b) or STARSadenovirus (c-h). Anti STARS antibody does not detect GFP protein norendogenous STARS (a). Overexpressed STARS is stained with anti-STARSantibody followed by fluoresceine anti-rabbit IgG secondary actibody (c,f). Z-band is stained with anti α-actinin antibody followed by Texas redconjugated secondary antibody (b, d). STARS was localized in thesarcomere (c and f), but not at the Z-band, as demonstrated by the lackof overlap with α-actinin (d, e). Phaloidin-TRITC shows the thinfilaments (g). STARS superimposes with the thin filament, but not atZ-lband (h). Bars: 10 μm (e, a-e in the same magnification), 5 μm (h)

[0031]FIG. 4—Deletion mapping of the actin-binding domain of STARS. FIG.4A: COS cells were transiently transfected with an expression vectorencoding STARS (wt) or deletion mutants with an N-terminal Myc-tag. Thesubcellular distribution of STARS was determined by immunofluorescenceusing fluoresceine anti-mouse IgG secondary antibody (c, f, i and l).Cells were also stained with Phalloidin-TRITC to visualize F-actin (a,b, d, g, j and m). F-actin and stress fibers were in COS cellstransfected with an empty vector (a, b). STARS (wt), shows the exactsame pattern of phalloidin staining; strongly associates with F-actinand induces extremely thick bundles (c, d, e). Numerous tumble stressfibers are also induced. (f, g, h). Homologous region C142 induces actinbundles (i, j, k). In contraast, homologous reigon deletion mutant C96just associates with actin stress fibers (1, m, n). Bar indicates 20 μm.FIG. 4B: COS cells were transiently transfected with an expressionvector encoding STARS or the indicated deletion mutants with Myc-epitopetags. Cell extracts were then immunoprecipitated with a rabbitpolyclonal anti-Myc antibody and immunoprecipitates were analyzed byWestern blot using anti-actin monoclonal antibody (top) or anti-Mycmonoclonal antibody (bottom).

[0032]FIG. 5—Summary of STARS deletion mutants. STARS (wt) and alldeletion mutants associates with actin stress fibers inimmunocytochemistry, Wild type STARS and deletion mutants (C142,Del234-247, Del248-262 and Del263-279) have an ability to induce actinbundles and activate SRF transcription.

[0033]FIG. 6—STARS stimulates SRF activity through a Rho-dependentmechanism. FIG. 6A: COS and F9 cells were transiently transfected withan SM22-luciferase reporter or the same reporter containing mutations inthe CArG-boxes within the SM22 promoter (mutSM22), and an STARSexpression vector. FIG. 6B: COS cells were transiently transfected withthe indicated reporter plasmids with and without an STARS expressionplasmid. FIG. 6C: COS cells were transiently transfected withSM22-luciferase and expression plasmids encoding the indicated STARSmutants. FIG. 6D: COS cells were transiently transfected withSM22-luciferase in the presence and absence of STARS and the indicatedagents. FIG. 6E: COS cells were transiently transfected withSM22-luciferase and a STARS or RhoA (L63) expression vector. FIG. 6F:COS cells were transiently transfected with SM22-luciferase and a STARSexpression vector in the presence of Y-27632 or a C3 expression vector.Luciferase activity was determined on cell extracts and is expressed asthe level of activity relative to that in cells transfected with theindicated reporters without STARS.

[0034]FIG. 7—A model for the involvement of STARS in actin dynamics andsignaling to SRF. The distribution of actin between the polymerized (Factin) and monomeri (G actin) states is determined by actintreadmilling. Actin monomers are added to the plus end and are removedfrom the minus end of actin filaments. STARS promotes the formation of Factin and activates Rho, which also promotes actin polymerization. Rhoactivates ROCK and other effectors leading to the stimulation of SRFactivity.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0035] Heart disease and its manifestations, including coronary arterydisease, myocardial infarction, congestive heart failure and cardiachypertrophy, presents a major health risk in the United States today.The cost to diagnose, treat and support patients suffering from thesediseases is well into the billions of dollars. Two particularly severemanifestations of heart disease are myocardial infarction and cardiachypertrophy. With respect to myocardial infarction, typically an acutethrombotic coronary occlusion occurs in a coronary artery as a result ofatherosclerosis and causes myocardial cell death. Becausecardiomyocytes, the heart muscle cells, are terminally differentiatedand generally incapable of cell division, they are generally replaced byscar tissue when they die during the course of an acute myocardialinfarction. Scar tissue is not contractile, fails to contribute tocardiac function, and often plays a detrimental role in heart functionby expanding during cardiac contraction, or by increasing the size andeffective radius of the ventricle, for example, becoming hypertrophic.With respect to cardiac hypertrophy, one theory regards this as adisease that resembles aberrant development and, as such, raises thequestion of whether developmental signals in the heart can contribute tohypertrophic disease.

[0036] The inventors have described herein a novel actin-binding proteindesignated as STARS (striated muscle activator of Rho signaling), whichis expressed specifically in cardiac and skeletal muscle cells and isupregulated in response to calcineurin signaling during cardiachypertrophy. STARS is identified in a differential cDNA screen forunknown genes expressed in the early embryonic heart. It is localized tothe thin filament of the sarcomere and to actin stress fibers where itpromotes actin bundling. STARS stimulates the transcriptional activityof SRF through a mechanism that requires actin bundling and Rho kinaseactivation. STARS provides a mechanism for selectively enhancing thetranscriptional activity of SRF in muscle cells and for linking changesin actin dynamics to gene transcription. These findings indicate thatSTARS acts as a muscle-specific transducer of cytoskeletal signals thatstimulate SRF activity.

[0037] I. STARS Peptides and Polypeptides

[0038] STARS is a novel and evolutionarily conservedactin-binding/bundling protein expressed specifically in striatedmuscle. STARS localizes to the thin filament of the sarcomere and toactin stress fibers. Stabilization of the actin cytoskeleton by STARSstimulates SRF-dependent transcription through a mechanism that involvesRhoA signaling.

[0039] STARS contains no recognizable protein motifs and represents anew type of actin-binding protein. Deletion mutants of STARS indicatethat there are multiple, nonoverlapping regions of the protein that canmediate association with actin stress fibers in vivo. However, only theconserved carboxy-terminal domain bundles F-actin and binds actin withsufficient affinity to be detect in co-immunoprecipitation assays. Thehigh conservation of this region between vertebrate and invertebrateproteins indicates that this activity has been evolutionarily conserved.It should also be noted that STARS associates with actin of thethin-filament, but not the Z-line. This raises the possibility that theSTARS-binding region of actin may be masked on the Z-line, possiblybecause of numerous other actin-binding proteins localized to thisstructure (Bang et al., 2001).

[0040] STARS expression is initiated in cardiac and skeletal muscleduring the period of myofibrillogenesis when the myofibrillar componentsbecome assembled into the functional sarcomere (Ehler et al., 1999;Gregorio and Antin, 2000). Considering the timing of its expression andits actin-binding properties, STARS may participate in sarcomereassembly by inducing actin polymerization and cross-linking duringstriated muscle development.

[0041] Consistent with recent studies implicating actin dynamics in thecontrol of SRF dependent transcription (Sotiropoulos et al., 1999; Weiet al., 2001; Mack et al., 2001), STARS stimulates the activity of SRF.The conserved carboxy-terminal region of STARS is both necessary andsufficient for SRF activation and actin bundling; the correlationbetween these activities argues that the effects of STARS on SRF arecoupled to its effects on the cytoskeleton. Stimulation of SRF activityis observed with native muscle promoters, as well as with an artificialpromoter containing multimerized CArG-boxes. However, STARS does notactivate all SRF-dependent promoters equally, which indicates thatadditional promoter-specific transcription factors modulate theresponsiveness of SRF to STARS-dependent signaling.

[0042] In addition to an entire STARS molecule, the present inventionalso relates to fragments of the polypeptides that may or may not retainseveral of the functions described below. Fragments, including theN-terminus of the molecule, may be generated by genetic engineering oftranslation stop sites within the coding region (discussed below).Alternatively, treatment of the STARS with proteolytic enzymes, known asproteases, can produce a variety of N-terminal, C-terminal and internalfragments. Examples of fragments may include contiguous residues of SEQID NOS:2, 4, 6, 8, and 10 of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80,85, 90, 95, 100, 200, 300, 400 or more amino acids in length. Thesefragments may be purified according to known methods, such asprecipitation (e.g., ammonium sulfate), HPLC, ion exchangechromatography, affinity chromatography (including immunoaffinitychromatography) or various size separations (sedimentation, gelelectrophoresis, gel filtration).

[0043] A. Variants of STARS

[0044] Amino acid sequence variants of the polypeptide can besubstitutional, insertional or deletion variants. Deletion variants lackone or more residues of the native protein that are not essential forfunction or immunogenic activity, and are exemplified by the variantslacking a transmembrane sequence described above. Another common type ofdeletion variant is one lacking secretory signal sequences or signalsequences directing a protein to bind to a particular part of a cell.Insertional mutants typically involve the addition of material at anon-terminal point in the polypeptide. This may include the insertion ofan immunoreactive epitope or simply a single residue. Terminaladditions, called fusion proteins, are discussed below.

[0045] Substitutional variants typically contain the exchange of oneamino acid for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptide, suchas stability against proteolytic cleavage, without the loss of otherfunctions or properties. Substitutions of this kind preferably areconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

[0046] The following is a discussion based upon changing of the aminoacids of a protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andits underlying DNA coding sequence, and nevertheless obtain a proteinwith like properties. It is thus contemplated by the inventors thatvarious changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

[0047] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

[0048] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte andDoolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0049] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e., still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0050] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5+1); alanine (−0.5);histidine*−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); tryptophan (−3.4).

[0051] It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent and immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin 2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

[0052] As outlined above, amino acid substitutions are generally basedon the relative similarity of the amino acid side-chain substituents,for example, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take several of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

[0053] Another embodiment for the preparation of polypeptides accordingto the invention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure (Johnson et al., 1993). The underlying rationale behind theuse of peptide mimetics is that the peptide backbone of proteins existschiefly to orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule. These principles may be used, in conjunction with theprinciples outline above, to engineer second generation molecules havingmany of the natural properties of STARS, but with altered and evenimproved characteristics.

[0054] B. Domain Switching

[0055] Domain switching involves the generation of chimeric moleculesusing different but, in this case, related polypeptides. These moleculesmay have additional value in that these “chimeras” can be distinguishedfrom natural molecules, while possibly providing the same function. Forexample, any of the various homologs from other species provide suitablecandidates for domain switching experiments.

[0056] C. Fusion Proteins

[0057] A specialized kind of insertional variant is the fusion protein.This molecule generally has all or a substantial portion of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide. For example, fusions typically employ leadersequences from other species to permit the recombinant expression of aprotein in a heterologous host. Another useful fusion includes theaddition of an immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions.

[0058] D. Purification of Proteins

[0059] It will be desirable to purify STARS or variants thereof. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the crude fractionation of thecellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography, polyacrylamide gel electrophoresis, andisoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

[0060] Certain aspects of the present invention concern thepurification, and in particular embodiments, the substantialpurification, of an encoded protein or peptide. The term “purifiedprotein or peptide” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the protein orpeptide is purified to any degree relative to its naturally-obtainablestate. A purified protein or peptide therefore also refers to a proteinor peptide, free from the environment in which it may naturally occur.

[0061] Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

[0062] Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

[0063] Various techniques suitable for use in protein purification willbe well known to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

[0064] There is no general requirement that the protein or peptidealways be provided in their most purified state. Indeed, it iscontemplated that less substantially purified products will have utilityin certain embodiments. Partial purification may be accomplished byusing fewer purification steps in combination, or by utilizing differentforms of the same general purification scheme. For example, it isappreciated that a cation-exchange column chromatography performedutilizing an HPLC apparatus will generally result in a greater “-fold”purification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of protein product,or in maintaining the activity of an expressed protein.

[0065] It is known that the migration of a polypeptide can vary,sometimes significantly, with different conditions of SDS/PAGE (Capaldiet al., 1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

[0066] High Performance Liquid Chromatography (HPLC) is characterized bya very rapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

[0067] Gel chromatography, or molecular sieve chromatography, is aspecial type of partition chromatography that is based on molecularsize. The theory behind gel chromatography is that the column, which isprepared with tiny particles of an inert substance that contain smallpores, separates larger molecules from smaller molecules as they passthrough or around the pores, depending on their size. As long as thematerial of which the particles are made does not adsorb the molecules,the sole factor determining rate of flow is the size. Hence, moleculesare eluted from the column in decreasing size, so long as the shape isrelatively constant. Gel chromatography is unsurpassed for separatingmolecules of different size because separation is independent of allother factors such as pH, ionic strength, temperature, etc. There alsois virtually no adsorption, less zone spreading and the elution volumeis related in a simple matter to molecular weight.

[0068] Affinity Chromatography is a chromatographic procedure thatrelies on the specific affinity between a substance to be isolated and amolecule that it can specifically bind to. This is a receptor-ligandtype interaction. The column material is synthesized by covalentlycoupling one of the binding partners to an insoluble matrix. The columnmaterial is then able to specifically adsorb the substance from thesolution. Elution occurs by changing the conditions to those in whichbinding will not occur (alter pH, ionic strength, temperature, etc.).

[0069] A particular type of affinity chromatography useful in thepurification of carbohydrate containing compounds is lectin affinitychromatography. Lectins are a class of substances that bind to a varietyof polysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

[0070] The matrix should be a substance that itself does not adsorbmolecules to any significant extent and that has a broad range ofchemical, physical and thermal stability. The ligand should be coupledin such a way as to not affect its binding properties. The ligand shouldalso provide relatively tight binding. And it should be possible toelute the substance without destroying the sample or the ligand. One ofthe most common forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

[0071] E. Synthetic Peptides

[0072] The present invention also describes smaller STARS-relatedpeptides for use in various embodiments of the present invention.Because of their relatively small size, the peptides of the inventioncan also be synthesized in solution or on a solid support in accordancewith conventional techniques. Various automatic synthesizers arecommercially available and can be used in accordance with knownprotocols. See, for example, Stewart and Young (1984); Tam et al.(1983); Merrifield (1986); and Barany and Merrifield (1979), eachincorporated herein by reference. Short peptide sequences, or librariesof overlapping peptides, usually from about 6 up to about 35 to 50 aminoacids, which correspond to the selected regions described herein, can bereadily synthesized and then screened in screening assays designed toidentify reactive peptides. Alternatively, recombinant DNA technologymay be employed wherein a nucleotide sequence which encodes a peptide ofthe invention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

[0073] F. Antigen Compositions

[0074] The present invention also provides for the use of STARS proteinsor peptides as antigens for the immunization of animals relating to theproduction of antibodies. It is envisioned that STARS, or portionsthereof, will be coupled, bonded, bound, conjugated or chemically-linkedto one or more agents via linkers, polylinkers or derivatized aminoacids. This may be performed such that a bispecific or multivalentcomposition or vaccine is produced. It is further envisioned that themethods used in the preparation of these compositions will be familiarto those of skill in the art and should be suitable for administrationto animals, i.e., pharmaceutically acceptable. Preferred agents as thecarriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin(BSA).

[0075] II. Nucleic Acids

[0076] The present invention also provides, in another embodiment, genesencoding STARS. Genes for mouse, human, zebrafish, Drosophila or c.elegans have been identified. See, for example, SEQ ID NOS: 1, 3, 5, 7and 9 respectively. The present invention is not limited in scope tothese genes, however, as one of ordinary skill in the art could, usingthese nucleic acids, readily identify related homologs in these andvarious other species (e.g., rat, rabbit, dog, monkey, gibbon, human,chimp, ape, baboon, cow, pig, horse, sheep, cat and other species).

[0077] In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, a “STARS gene” may contain a variety of different bases and yetstill produce a corresponding polypeptide that is functionallyindistinguishable, and in some cases structurally, from the human andmouse genes disclosed herein.

[0078] Similarly, any reference to a nucleic acid should be read asencompassing a host cell containing that nucleic acid and, in somecases, capable of expressing the product of that nucleic acid. Inaddition to therapeutic considerations, cells expressing nucleic acidsof the present invention may prove useful in the context of screeningfor agents that induce, repress, inhibit, augment, interfere with,block, abrogate, stimulate or enhance the activity of STARS.

[0079] A. Nucleic Acids Encoding STARS

[0080] Nucleic acids according to the present invention may encode anentire STARS gene, a domain of STARS, or any other fragment of STARS asset forth herein. The nucleic acid may be derived from genomic DNA,i.e., cloned directly from the genome of a particular organism. Inpreferred embodiments, however, the nucleic acid would comprisecomplementary DNA (cDNA). Also contemplated is a cDNA plus a naturalintron or an intron derived from another gene; such engineered moleculesare sometime referred to as “mini-genes.” At a minimum, these and othernucleic acids of the present invention may be used as molecular weightstandards in, for example, gel electrophoresis.

[0081] The term “cDNA” is intended to refer to DNA prepared usingmessenger RNA (mRNA) as template. The advantage of using a cDNA, asopposed to genomic DNA or DNA polymerized from a genomic, non- orpartially-processed RNA template, is that the cDNA primarily containscoding sequences of the corresponding protein. There may be times whenthe full or partial genomic sequence is preferred, such as where thenon-coding regions are required for optimal expression or wherenon-coding regions such as introns are to be targeted in an antisensestrategy.

[0082] It also is contemplated that a given STARS from a given speciesmay be represented by natural variants that have slightly differentnucleic acid sequences but, nonetheless, encode the same protein (seeTable 1 below).

[0083] As used in this application, the term “a nucleic acid encoding aSTARS” refers to a nucleic acid molecule that has been isolated free oftotal cellular nucleic acid. In preferred embodiments, the inventionconcerns a nucleic acid sequence essentially as set forth in SEQ ID NOS:1, 3, 5, 7 or 9 (mouse, human, zebrafish, Drosophila or c. elegansrespectively). The term “as set forth in SEQ ID NOS: 1 or 3, 5, 7 or 9”means that the nucleic acid sequence substantially corresponds to aportion of SEQ ID NO:1 or 3, 5, 7 or 9. The term “functionallyequivalent codon” is used herein to refer to codons that encode the sameamino acid, such as the six codons for arginine or serine (Table 1,below), and also refers to codons that encode biologically equivalentamino acids, as discussed in the following pages. TABLE 1 Amino AcidsCodons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Asparticacid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUCUUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine IleI AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0084] Allowing for the degeneracy of the genetic code, sequences thathave at least about 50%, usually at least about 60%, more usually about70%, most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of SEQ ID NOS:1 or 3, 5, 7 or 9 are contemplated. Sequencesthat are essentially the same as those set forth in SEQ ID NOS:1, 3, 5,7 or 9 may also be functionally defined as sequences that are capable ofhybridizing to a nucleic acid segment containing the complement of SEQID NOS:1, 3, 5, 7 or 9 under standard conditions.

[0085] The DNA segments of the present invention include those encodingbiologically functional equivalent STARS proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

[0086] B. Oligonucleotide Probes and Primers

[0087] Naturally, the present invention also encompasses DNA segmentsthat are complementary, or essentially complementary, to the sequenceset forth in SEQ ID NOS:1, 3, 5, 7 or 9. Nucleic acid sequences that are“complementary” are those that are capable of base-pairing according tothe standard Watson-Crick complementary rules. As used herein, the term“complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NOS:1, 3, 5, 7 or 9under relatively stringent conditions such as those described herein.Such sequences may encode entire STARS proteins or functional ornon-functional fragments thereof.

[0088] Alternatively, the hybridizing segments may be shorteroligonucleotides. Sequences of 17 bases long should occur only once inthe human genome and, therefore, suffice to specify a unique targetsequence. Although shorter oligomers are easier to make and increase invivo accessibility, numerous other factors are involved in determiningthe specificity of hybridization. Both binding affinity and sequencespecificity of an oligonucleotide to its complementary target increaseswith increasing length. It is contemplated that exemplaryoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morebase pairs will be used, although others are contemplated. Longerpolynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or5000 bases and longer are contemplated as well. Such oligonucleotideswill find use, for example, as probes in Southern and Northern blots andas primers in amplification reactions.

[0089] Suitable hybridization conditions will be well known to those ofskill in the art. In certain applications, for example, substitution ofamino acids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

[0090] In other embodiments, hybridization may be achieved underconditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mMMgCl₂, 10 mM dithiothreitol, at temperatures between approximately 20°C. to about 37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

[0091] One method of using probes and primers of the present inventionis in the search for genes related to STARS or, more particularly,homologs of STARS from other species. Normally, the target DNA will be agenomic or cDNA library, although screening may involve analysis of RNAmolecules. By varying the stringency of hybridization, and the region ofthe probe, different degrees of homology may be discovered.

[0092] Another way of exploiting probes and primers of the presentinvention is in site-directed, or site-specific mutagenesis.Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

[0093] The technique typically employs a bacteriophage vector thatexists in both a single-stranded and double-stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage. These phage vectors are commercially available and their useis generally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

[0094] In general, site-directed mutagenesis is performed by firstobtaining a single-stranded vector, or melting of two strands of adouble-stranded vector which includes within its sequence a DNA sequenceencoding the desired protein. An oligonucleotide primer bearing thedesired mutated sequence is synthetically prepared. This primer is thenannealed with the single-stranded DNA preparation, taking into accountthe degree of mismatch when selecting hybridization conditions, andsubjected to DNA polymerizing enzymes such as E. coli polymerase IKlenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform appropriate cells, such as E. coli cells, and clones areselected that include recombinant vectors bearing the mutated sequencearrangement.

[0095] The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

[0096] C. Antisense Constructs

[0097] Antisense methodology takes advantage of the fact that nucleicacids tend to pair with “complementary” sequences. By complementary, itis meant that polynucleotides are those which are capable ofbase-pairing according to the standard Watson-Crick complementarityrules. That is, the larger purines will base pair with the smallerpyrimidines to form combinations of guanine paired with cytosine (G:C)and adenine paired with either thymine (A:T) in the case of DNA, oradenine paired with uracil (A:U) in the case of RNA. Inclusion of lesscommon bases such as inosine, 5-methylcytosine, 6-methyladenine,hypoxanthine and others in hybridizing sequences does not interfere withpairing.

[0098] Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

[0099] Antisense constructs may be designed to bind to the promoter andother control regions, exons, introns or even exon-intron boundaries ofa gene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

[0100] As stated above, “complementary” or “antisense” meanspolynucleotide sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, sequencesof fifteen bases in length may be termed complementary when they havecomplementary nucleotides at thirteen or fourteen positions. Naturally,sequences which are completely complementary will be sequences which areentirely complementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

[0101] It may be advantageous to combine portions of genomic DNA withcDNA or synthetic sequences to generate specific constructs. Forexample, where an intron is desired in the ultimate construct, a genomicclone will need to be used. The cDNA or a synthesized polynucleotide mayprovide more convenient restriction sites for the remaining portion ofthe construct and, therefore, would be used for the rest of thesequence.

[0102] D. Ribozymes

[0103] Although proteins traditionally have been used for catalysis ofnucleic acids, another class of macromolecules has emerged as useful inthis endeavor. Ribozymes are RNA-protein complexes that cleave nucleicacids in a site-specific fashion. Ribozymes have specific catalyticdomains that possess endonuclease activity (Kim and Cook, 1987; Gerlachet al., 1987; Forster and Symons, 1987). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cook et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

[0104] Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cook et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., 1991; Sarver et al.,1990). Recently, it was reported that ribozymes elicited genetic changesin some cells lines to which they were applied; the altered genesincluded the oncogenes H-ras, c-fos and genes of HIV. Most of this workinvolved the modification of a target mRNA, based on a specific mutantcodon that is cleaved by a specific ribozyme.

[0105] E. Vectors for Cloning, Gene Transfer and Expression

[0106] Within certain embodiments expression vectors are employed toexpress a STARS polypeptide product, which can then be purified and, forexample, be used to vaccinate animals to generate antisera or monoclonalantibody with which further studies may be conducted. In otherembodiments, the expression vectors are used in gene therapy. Expressionrequires that appropriate signals be provided in the vectors, and whichinclude various regulatory elements, such as enhancers/promoters fromboth viral and mammalian sources that drive expression of the genes ofinterest in host cells. Elements designed to optimize messenger RNAstability and translatability in host cells also are defined. Theconditions for the use of a number of dominant drug selection markersfor establishing permanent, stable cell clones expressing the productsare also provided, as is an element that links expression of the drugselection markers to expression of the polypeptide.

[0107] (i) Regulatory Elements

[0108] Throughout this application, the term “expression construct” ismeant to include any type of genetic construct containing a nucleic acidcoding for a gene product in which part or all of the nucleic acidencoding sequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

[0109] In preferred embodiments, the nucleic acid encoding a geneproduct is under transcriptional control of a promoter. A “promoter”refers to a DNA sequence recognized by the synthetic machinery of thecell, or introduced synthetic machinery, required to initiate thespecific transcription of a gene. The phrase “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene.

[0110] The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

[0111] At least one module in each promoter functions to position theSTARSt site for RNA synthesis. The best known example of this is theTATA box, but in some promoters lacking a TATA box, such as the promoterfor the mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying theSTARSt site itself helps to fix the place of initiation.

[0112] Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the STARSt site, although a number of promotershave recently been shown to contain functional elements downstream ofthe STARSt site as well. The spacing between promoter elementsfrequently is flexible, so that promoter function is preserved whenelements are inverted or moved relative to one another. In the tkpromoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either co-operativelyor independently to activate transcription.

[0113] In various embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter, the Rous sarcoma viruslong terminal repeat, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose.

[0114] By employing a promoter with well-known properties, the level andpattern of expression of the protein of interest following transfectionor transformation can be optimized. Further, selection of a promoterthat is regulated in response to specific physiologic signals can permitinducible expression of the gene product. Tables 2 and 3 list severalregulatory elements that may be employed, in the context of the presentinvention, to regulate the expression of the gene of interest. This listis not intended to be exhaustive of all the possible elements involvedin the promotion of gene expression but, merely, to be exemplarythereof.

[0115] Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

[0116] The basic distinction between enhancers and promoters isoperational. An enhancer region as a whole must be able to stimulatetranscription at a distance; this need not be true of a promoter regionor its component elements. On the other hand, a promoter must have oneor more elements that direct initiation of RNA synthesis at a particularsite and in a particular orientation, whereas enhancers lack thesespecificities. Promoters and enhancers are often overlapping andcontiguous, often seeming to have a very similar modular organization.

[0117] Below is a list of viral promoters, cellular promoters/enhancersand inducible promoters/enhancers that could be used in combination withthe nucleic acid encoding a gene of interest in an expression construct(Table 2 and Table 3). Additionally, any other promoter/enhancercombination (for example, as per the Eukaryotic Promoter Data Base EPDB)could also be used to drive expression of the gene. Eukaryotic cells cansupport cytoplasmic transcription from certain bacterial promoters ifthe appropriate bacterial polymerase is provided, either as part of thedelivery complex or as an additional genetic expression construct. TABLE2 Promoter and/or Enhancer Promoter/Enhancer References ImmunoglobulinHeavy Chain Banerji et al., 1983; Gilles et al., 1983; Grosschedl etal., 1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinbergeret al., 1984; Kiledijan et al., 1988; Porton et al.; 1990 ImmunoglobulinLight Chain Queen et al., 1983; Picard et al., 1984 T-Cell ReceptorLuria et al., 1987; Winoto et al., 1989; Redondo et al.; 1990 HLA DQ aand/or DQ β Sullivan et al., 1987 β-Interferon Goodbourn et al., 1986;Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2 Green et al.,1989 Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHCClass II 5 Koch et al., 1989 MHC Class II HLA-DRa Sherman et al., 1989β-Actin Kawamoto et al., 1988; Mg et al.; 1989 Muscle Creatine Kinase(MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnson et al., 1989Prealbumin (Transthyretin) Costa et al., 1988 Elastase I Ornitz et al.,1987 Metallothionein (MTII) Karin et al., 1987; Culotta et al., 1989Collagenase Pinkert et al., 1987; Angel et al., 1987a Albumin Pinkert etal., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godbout et al.,1988; Campere et al., 1989 t-Globin Bodine et al., 1987; Perez-Stable etal., 1990 β-Globin Trudel et al., 1987 c-fos Cohen et al., 1987 c-HA-rasTriesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM) α₁-AntitrypainLatimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse and/orType I Collagen Ripe et al., 1989 Glucose-Regulated Proteins Change etal., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et al., 1986 HumanSerum Amyloid A (SAA) Edbrooke et al., 1989 Troponin I (TN I) Yutzey etal., 1989 Platelet-Derived Growth Factor Pech et al., 1989 (PDGF)Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerji et al.,1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herret al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al.,1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka etal., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villierset al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell and/orVillarreal, 1988 Retroviruses Kriegler et al., 1982, 1983; Levinson etal., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986;Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988;Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989 PapillomaVirus Campo et al., 1983; Lusky et al., 1983; Spandidos and/or Wilkie,1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987;Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987; Glueet al., 1988 Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986;Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988 HumanImmunodeficiency Virus Muessing et al., 1987; Hauber et al., 1988;Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosenet al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al.,1989; Braddock et al., 1989 Cytomegalovirus (CMV) Weber et al., 1984;Boshart et al., 1985; Foecking et al., 1986 Gibbon Ape Leukemia VirusHolbrook et al., 1987; Quinn et al., 1989

[0118] TABLE 3 Inducible Elements Element Inducer References MT IIPhorbol Ester (TFA) Palmiter et al., 1982; Heavy metals Haslinger etal., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al.,1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV(mouse Glucocorticoids Huang et al., 1991; Lee et mammary al., 1981;Majors et al., tumor virus) 1983; Chandler et al., 1983; Lee et al.,1984; Ponta et al., 1985; Sakai et al., 1988 β-Interferon poly(rI)xTavernier et al., 1983 poly(rc) Adenovirus 5 E2 E1A Imperiale et al.,1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a StromelysinPhorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angelet al., 1987b Murine MX Gene Interferon, Newcastle Hug et al., 1988Disease Virus GRP78 Gene A23187 Resendez et al., 1988 α-2-MacroglobulinIL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class IInterferon Blanar et al., 1989 Gene H-2κb HSP70 E1A, SV40 Large T Tayloret al., 1989, 1990a, Antigen 1990b Proliferin Phorbol Ester-TPA Mordacqet al., 1989 Tumor Necrosis PMA Hensel et al., 1989 Factor ThyroidStimulating Thyroid Hormone Chatterjee et al., 1989 Hormone α Gene

[0119] Of particular interest are muscle specific promoters, and moreparticularly, cardiac specific promoters. These include the myosin lightchain-2 promoter (Franz et al., 1994; Kelly et al., 1995), the α actinpromoter (Moss et al., 1996), the troponin 1 promoter (Bhavsar et al.,1996); the Na⁺/Ca²⁺ exchanger promoter (Barnes et al., 1997), thedystrophin promoter (Kimura et al., 1997), the creatine kinase promoter(Ritchie, M. E., 1996), the α7 integrin promoter (Ziober & Kramer,1996), the brain natriuretic peptide promoter (LaPointe et al., 1996),the α B-crystallin/small heat shock protein promoter (Gopal-Srivastava,R., 1995), and a myosin heavy chain promoter (Yamauchi-Takihara et al.,1989) and the ANF promoter (LaPointe et al., 1988).

[0120] Where a cDNA insert is employed, one will typically desire toinclude a polyadenylation signal to effect proper polyadenylation of thegene transcript. The nature of the polyadenylation signal is notbelieved to be crucial to the successful practice of the invention, andany such sequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

[0121] (ii) Selectable Markers

[0122] In certain embodiments of the invention, the cells containnucleic acid constructs of the present invention, a cell may beidentified in vitro or in vivo by including a marker in the expressionconstruct. Such markers would confer an identifiable change to the cellpermitting easy identification of cells containing the expressionconstruct. Usually the inclusion of a drug selection marker aids incloning and in the selection of transformants, for example, genes thatconfer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocinand histidinol are useful selectable markers. Alternatively, enzymessuch as herpes simplex virus thymidine kinase (tk) or chloramphenicolacetyltransferase (CAT) may be employed. Immunologic markers also can beemployed. The selectable marker employed is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable markers are well known to one of skill in the art.

[0123] (iii) Multigene Constructs and IRES

[0124] In certain embodiments of the invention, the use of internalribosome binding sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornaovirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

[0125] Any heterologous open reading frame can be linked to IRESelements. This includes genes for secreted proteins, multi-subunitproteins, encoded by independent genes, intracellular or membrane-boundproteins and selectable markers. In this way, expression of severalproteins can be simultaneously engineered into a cell with a singleconstruct and a single selectable marker.

[0126] (iv) Delivery of Expression Constructs

[0127] There are a number of ways in which expression constructs may beintroduced into cells. In certain embodiments of the invention, a vector(also referred to herein as a gene delivery vector) is employed todeliver the expression construct. By way of illustration, in someembodiments, the vector comprises a virus or engineered constructderived from a viral genome. The ability of certain viruses to entercells via receptor-mediated endocytosis, to integrate into host cellgenome and express viral genes stably and efficiently have made themattractive candidates for the transfer of foreign genes into mammaliancells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal andSugden, 1986; Temin, 1986). The first viruses used as gene deliveryvectors were DNA viruses including the papovaviruses (simian virus 40,bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal andSugden, 1986). Generally, these have a relatively low capacity forforeign DNA sequences and have a restricted host spectrum. They canaccommodate only up to 8 kb of foreign genetic material but can bereadily introduced in a variety of cell lines and laboratory animals(Nicolas and Rubenstein, 1988; Temin, 1986). Where viral vectors areemployed to deliver the gene or genes of interest, it is generallypreferred that they be replication-defective, for example as known tothose of skill in the art and as described further herein below.

[0128] One of the preferred methods for in vivo delivery of expressionconstructs involves the use of an adenovirus expression vector.“Adenovirus expression vector” is meant to include those constructscontaining adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to express a polynucleotide that has been clonedtherein. In this context, expression does not require that the geneproduct be synthesized.

[0129] In preferred embodiments, the expression vector comprises agenetically engineered form of adenovirus. Knowledge of the geneticorganization of adenovirus, a 36 kb, linear, double-stranded DNA virus,allows substitution of large pieces of adenoviral DNA with foreignsequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast toretrovirus, the adenoviral infection of host cells does not result inchromosomal integration because adenoviral DNA can replicate in anepisomal manner without potential genotoxicity. Also, adenoviruses arestructurally stable, and no genome rearrangement has been detected afterextensive amplification. Adenovirus can infect virtually all epithelialcells regardless of their cell cycle stage and are able to infectnon-dividing cells such as, for example, cardiomyocytes. So far,adenoviral infection appears to be linked only to mild disease such asacute respiratory disease in humans.

[0130] Adenovirus is particularly suitable for use as a gene deliveryvector because of its mid-sized genome, ease of manipulation, hightiter, wide target cell range and high infectivity. Both ends of theviral genome contain 100-200 base pair inverted repeats (ITRs), whichare cis elements necessary for viral DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression and host cellshut-off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP, (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNA's issued from this promoter possess a 5′-tripartite leader (TPL)sequence which makes them preferred mRNA's for translation.

[0131] In a current system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it isimportant to minimize this possibility by, for example, reducing oreliminating adnoviral sequence overlaps within the system and/or toisolate a single clone of virus from an individual plaque and examineits genomic structure.

[0132] Generation and propagation of the current adenovirus vectors,which are replication deficient, depend on a unique helper cell line,designated 293, which was transformed from human embryonic kidney cellsby Ad5 DNA fragments and constitutively expresses E1 proteins (Graham etal., 1977). Since the E3 region is dispensable from the adenovirusgenome (Jones and Shenk, 1978), the current adenovirus vectors, with thehelp of 293 cells, carry foreign DNA in either the E1, the E3 or bothregions (Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of such adenovirus vectors is about 7.5kb, or about 15% of the total length of the vector. Additionally,modified adenoviral vectors are now available which have an even greatercapacity to carry foreign DNA.

[0133] Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,a preferred helper cell line is 293.

[0134] Racher et al. (1995) disclosed improved methods for culturing 293cells and propagating adenovirus. In one format, natural cell aggregatesare grown by inoculating individual cells into 1 liter siliconizedspinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for 1to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

[0135] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be selected from any ofthe 42 different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is a preferred STARSting material for obtaining areplication-defective adenovirus vector for use in the presentinvention. This is, in part, because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

[0136] As stated above, a preferred adenoviral vector according to thepresent invention lacks an adenovirus E1 region and thus, isreplication. Typically, it is most convenient to introduce thepolynucleotide encoding the gene of interest at the position from whichthe E1-coding sequences have been removed. However, the position ofinsertion of the construct within the adenovirus sequences is notcritical to the invention. Further, other adenoviral sequences may bedeleted and/or inactivated in addition to or in lieu of the E1 region.For example, the E2 and E4 regions are both necessary for adenoviralreplication and thus may be modified to render an adenovirus vectorreplication-defective, in which case a helper cell line or helper viruscomplex may employed to provide such deleted/inactivated genes in trans.The polynucleotide encoding the gene of interest may alternatively beinserted in lieu of a deleted E3 region such as in E3 replacementvectors as described by Karlsson et al. (1986), or in a deleted E4region where a helper cell line or helper virus complements the E4defect. Other modifications are known to those of skill in the art andare likewise contemplated herein.

[0137] Adenovirus is easy to grow and manipulate and exhibits broad hostrange in vitro and in vivo. This group of viruses can be obtained inhigh titers, e.g., 10⁹-10¹² plaque-forming units per ml, and they arehighly infective. The life cycle of adenovirus does not requireintegration into the host cell genome. The foreign genes delivered byadenovirus vectors are episomal and, therefore, have low genotoxicity tohost cells. No side effects have been reported in studies of vaccinationwith wild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

[0138] Adenovirus vectors have been used in eukaryotic gene expression(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development(Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animalstudies indicated that recombinant adenovirus could be used for genetherapy (Stratford-Perricaudet and Perricaudet, 1991;Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies inadministering recombinant adenovirus to different tissues includeadministration via intracoronary catheter into one or more coronaryarteries of the heart (Hammond, et al., U.S. Pat. Nos. 5,792,453 and6,100,242) trachea instillation (Rosenfeld et al., 1991; Rosenfeld etal., 1992), muscle injection (Ragot et al., 1993), peripheralintravenous injections (Herz and Gerard, 1993) and stereotacticinoculation into the brain (Le Gal La Salle et al., 1993).

[0139] The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

[0140] In order to construct a retroviral vector, a nucleic acidencoding a gene of interest is inserted into the viral genome in theplace of certain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

[0141] A novel approach designed to allow specific targeting ofretrovirus vectors was recently developed based on the chemicalmodification of a retrovirus by the chemical addition of lactoseresidues to the viral envelope. This modification could permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

[0142] A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

[0143] There are certain limitations to the use of retrovirus vectors inall aspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which theintact-sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

[0144] Other viral vectors may be employed as expression constructs inthe present invention. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

[0145] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thisindicated that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al., recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas co-transfected with wild-type virus into an avian hepatoma cellline. Culture media containing high titers of the recombinant virus wereused to infect primary duckling hepatocytes. Stable CAT gene expressionwas detected for at least 24 days after transfection (Chang et al.,1991).

[0146] In order to effect expression of sense or antisense geneconstructs, the expression construct must be delivered into a cell. Thisdelivery may be accomplished in vitro, as in laboratory procedures fortransforming cells lines, or in vivo or ex vivo, as in the treatment ofcertain disease states. In general, viral vectors accomplish delivery ofthe expression construct by infecting the target cells of interest.Alternatively to incorporating the expression construct into the genomeof a viral vector, the expression construct may be encapsidated in theinfectious viral particle.

[0147] Several non-viral gene delivery vectors for the transfer ofexpression constructs into mammalian cells also are contemplated by thepresent invention. These include calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990)DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986;Potter et al., 1984), direct microinjection (Harland and Weintraub,1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al.,1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer etal., 1987), gene bombardment using high velocity microprojectiles (Yanget al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wuand Wu, 1988). Some of these techniques may be successfully adapted forin vivo or ex vivo use.

[0148] Once the expression construct has been delivered into the cellthe nucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

[0149] In yet another embodiment of the invention, the expression vectormay simply consist of naked recombinant DNA or plasmids comprising theexpression construct. Transfer of the construct may be performed by anyof the methods mentioned above which physically or chemicallypermeabilize the cell membrane. This is particularly applicable fortransfer in vitro but it may be applied to in vivo use as well. Dubenskyet al. (1984) successfully injected polyomavirus DNA in the form ofcalcium phosphate precipitates into liver and spleen of adult andnewborn mice demonstrating active viral replication and acute infection.Benvenisty and Neshif (1986) also demonstrated that directintraperitoneal injection of calcium phosphate-precipitated plasmidsresults in expression of the transfected genes. It is envisioned thatDNA encoding a gene of interest may also be transferred in a similarmanner in vivo and express the gene product.

[0150] In still another embodiment of the invention, transferring of anaked DNA expression construct into cells may involve particlebombardment. This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

[0151] Selected organs including the liver, skin, and muscle tissue ofrats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin etal., 1991). This may require surgical exposure of the tissue or cells,to eliminate any intervening tissue between the gun and the targetorgan, i.e., ex vivo treatment. Again, DNA encoding a particular genemay be delivered via this method and still be incorporated by thepresent invention.

[0152] In a further embodiment of the invention, the expressionconstruct may be entrapped in a liposome, another non-viral genedelivery vector. Liposomes are vesicular structures characterized by aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theyform spontaneously when phospholipids are suspended in an excess ofaqueous solution. The lipid components undergo self-rearrangement beforethe formation of closed structures and entrap water and dissolvedsolutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Alsocontemplated are lipofectamine-DNA complexes.

[0153] Liposome-mediated nucleic acid delivery and expression of foreignDNA in vitro has been very successful. Wong et al., (1980) demonstratedthe feasibility of liposome-mediated delivery and expression of foreignDNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

[0154] In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al, 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

[0155] Other expression constructs which can be employed to deliver anucleic acid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

[0156] Receptor-mediated gene targeting vehicles generally consist oftwo components: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0 273 085).

[0157] In other embodiments, the delivery vehicle may comprise a ligandand a liposome. For example, Nicolau et al. (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding aparticular gene also may be specifically delivered into a cell type byany number of receptor-ligand systems with or without liposomes. Forexample, epidermal growth factor (EGF) may be used as the receptor formediated delivery of a nucleic acid into cells that exhibit upregulationof EGF receptor. Mannose can be used to target the mannose receptor onliver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25(T-cell leukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

[0158] In certain embodiments, gene transfer may more easily beperformed under ex vivo conditions. Ex vivo gene therapy refers to theisolation of cells from an animal, the delivery of a nucleic acid intothe cells in vitro, and then the return of the modified cells back intoan animal. This may involve the surgical removal of tissue/organs froman animal or the primary culture of cells and tissues.

[0159] III. Generating Antibodies Reactive with STARS

[0160] In another aspect, the present invention contemplates an antibodythat is immunoreactive with a STARS molecule of the present invention,or any portion thereof. An antibody can be a polyclonal or a monoclonalantibody. In a preferred embodiment, an antibody is a monoclonalantibody. Means for preparing and characterizing antibodies are wellknown in the art (see, e.g., Harlow and Lane, 1988).

[0161] Briefly, a polyclonal antibody is prepared by immunizing ananimal with an immunogen comprising a polypeptide of the presentinvention and collecting antisera from that immunized animal. A widerange of animal species can be used for the production of antisera.Typically an animal used for production of anti-antisera is a non-humananimal including rabbits, mice, rats, hamsters, pigs or horses. Becauseof the relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

[0162] Antibodies, both polyclonal and monoclonal, specific for isoformsof antigen may be prepared using conventional immunization techniques,as will be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

[0163] It is proposed that the monoclonal antibodies of the presentinvention will find useful application in standard immunochemicalprocedures, such as ELISA and Western blot methods and inimmunohistochemical procedures such as tissue staining, as well as inother procedures which may utilize antibodies specific to STARS-relatedantigen epitopes. Additionally, it is proposed that monoclonalantibodies specific to the particular STARS of different species may beutilized in other useful applications

[0164] In general, both polyclonal and monoclonal antibodies againstSTARS may be used in a variety of embodiments. For example, they may beemployed in antibody cloning protocols to obtain cDNAs or genes encodingother STARS. They may also be used in inhibition studies to analyze theeffects of STARS related peptides in cells or animals. STARS antibodieswill also be useful in immunolocalization studies to analyze thedistribution of STARS during various cellular events, for example, todetermine the cellular or tissue-specific distribution of STARSpolypeptides under different points in the cell cycle. A particularlyuseful application of such antibodies is in purifying native orrecombinant STARS, for example, using an antibody affinity column. Theoperation of all such immunological techniques will be known to those ofskill in the art in light of the present disclosure.

[0165] Means for preparing and characterizing antibodies are well knownin the art (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare given in the examples below.

[0166] As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

[0167] As also is well known in the art, the immunogenicity of aparticular immunogen composition can be enhanced by the use ofnon-specific stimulators of the immune response, known as adjuvants.Exemplary and preferred adjuvants include complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

[0168] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster, injection mayalso be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate mAbs.

[0169] MAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified STARS protein, polypeptide orpeptide or cell expressing high levels of STARS. The immunizingcomposition is administered in a manner effective to stimulate antibodyproducing cells. Rodents such as mice and rats are preferred animals,however, the use of rabbit, sheep frog cells is also possible. The useof rats may provide certain advantages (Goding, 1986), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

[0170] Following immunization, somatic cells with the potential forproducing antibodies, specifically B-lymphocytes (B-cells), are selectedfor use in the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0171] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0172] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, 1986; Campbell, 1984). For example,where the immunized animal is a mouse, one may use P3-X63/Ag8,P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bu1; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with cell fusions.

[0173] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 2:1 ratio, though the ratio may vary from about20:1 to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

[0174] Fusion procedures usually produce viable hybrids at lowfrequencies, around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0175] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B-cells.

[0176] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

[0177] The selected hybridomas would then be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

[0178] IV. Diagnosing and Treating Defects in STARS

[0179] The inventors believe that STARS plays an important role in thedevelopment of cardiac tissue and, further, in the mechanisms of heartdisease. Thus, in another embodiment, there are provided methods fordiagnosing defects in STARS expression and function. More specifically,point mutations, deletions, insertions or regulatory pertubationsrelating to STARS, as well as increases or decrease in levels ofexpression, may be assessed using standard technologies, as describedbelow.

[0180] A. Genetic Diagnosis

[0181] One embodiment of the instant invention comprises a method fordetecting variation in the expression of STARS. This may comprisedetermining the level of STARS or determining specific alterations inthe expressed product.

[0182] A suitable biological sample can be any tissue or fluid. Variousembodiments include cells of the skin, muscle, facia, brain, prostate,breast, endometrium, lung, head & neck, pancreas, small intestine, bloodcells, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen,lymph node, bone marrow or kidney. Other embodiments include fluidsamples such as peripheral blood, lymph fluid, ascites, serous fluid,pleural effusion, sputum, cerebrospinal fluid, lacrimal fluid, stool orurine.

[0183] Nucleic acid used is isolated from cells contained in thebiological sample, according to standard methodologies (Sambrook et al.,1989). The nucleic acid may be genomic DNA or fractionated or whole cellRNA. Where RNA is used, it may be desired to convert the RNA to acomplementary DNA. In one embodiment, the RNA is whole cell RNA; inanother, it is poly-A RNA. Normally, the nucleic acid is amplified.

[0184] Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

[0185] Various types of defects may be identified by the presentmethods. Thus, “alterations” should be read as including deletions,insertions, point mutations and duplications. Point mutations result instop codons, frameshift mutations or amino acid substitutions. Somaticmutations are those occurring in non-germline tissues. Germ-linemutations can occur in any tissue and are inherited. Mutations in andoutside the coding region also may affect the amount of STARS produced,both by altering the transcription of the gene or in destabilizing orotherwise altering the processing of either the transcript (mRNA) orprotein.

[0186] It is contemplated that other mutations in the STARS genes may beidentified in accordance with the present invention. A variety ofdifferent assays are contemplated in this regard, including but notlimited to, fluorescent in situ hybridization (FISH), direct DNAsequencing, PFGE analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR™-SSCP.

[0187] (i) Primers and Probes

[0188] The term primer, as defined herein, is meant to encompass anynucleic acid that is capable of priming the synthesis of a nascentnucleic acid in a template-dependent process. Typically, primers areoligonucleotides from ten to twenty base pairs in length, but longersequences can be employed. Primers may be provided in double-stranded orsingle-stranded form, although the single-stranded form is preferred.Probes are defined differently, although they may act as primers.Probes, while perhaps capable of priming, are designed to binding to thetarget DNA or RNA and need not be used in an amplification process.

[0189] In preferred embodiments, the probes or primers are labeled withradioactive species (³²P, ¹⁴C, ³⁵S, ³H, or other label), with afluorophore (rhodamine, fluorescein) or a chemillumiscent (luciferase).

[0190] (ii) Template Dependent Amplification Methods

[0191] A number of template dependent processes are available to amplifythe marker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety.

[0192] Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

[0193] A reverse transcriptase PCR™ amplification procedure may beperformed in order to quantify the amount of mRNA amplified. Methods ofreverse transcribing RNA into cDNA are well known and described inSambrook et al., 1989. Alternative methods for reverse transcriptionutilize thermostable, RNA-dependent DNA polymerases. These methods aredescribed in WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

[0194] Another method for amplification is the ligase chain reaction(“LCR”), disclosed in EPO No. 320 308, incorporated herein by referencein its entirety. In LCR, two complementary probe pairs are prepared, andin the presence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

[0195] Methods based on ligation of two (or more) oligonucleotides inthe presence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. Wu etal., (1989), incorporated herein by reference in its entirety.

[0196] (iii) Southern/Northern Blotting

[0197] Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting or RNA species.

[0198] Briefly, a probe is used to target a DNA or RNA species that hasbeen immobilized on a suitable matrix, often a filter of nitrocellulose.The different species should be spatially separated to facilitateanalysis. This often is accomplished by gel electrophoresis of nucleicacid species followed by “blotting” on to the filter.

[0199] Subsequently, the blotted target is incubated with a probe(usually labeled) under conditions that promote denaturation andrehybridization. Because the probe is designed to base pair with thetarget, the probe will binding a portion of the target sequence underrenaturing conditions. Unbound probe is then removed, and detection isaccomplished as described above.

[0200] (iv) Separation Methods

[0201] It normally is desirable, at one stage or another, to separatethe amplification product from the template and the excess primer forthe purpose of determining whether specific amplification has occurred.In one embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

[0202] Alternatively, chromatographic techniques may be employed toeffect separation. There are many kinds of chromatography which may beused in the present invention: adsorption, partition, ion-exchange andmolecular sieve, and many specialized techniques for using themincluding column, paper, thin-layer and gas chromatography (Freifelder,1982).

[0203] (v) Detection Methods

[0204] Products may be visualized in order to confirm amplification ofthe marker sequences. One typical visualization method involves stainingof a gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

[0205] In one embodiment, visualization is achieved indirectly.Following separation of amplification products, a labeled nucleic acidprobe is brought into contact with the amplified marker sequence. Theprobe preferably is conjugated to a chromophore but may be radiolabeled.In another embodiment, the probe is conjugated to a binding partner,such as an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

[0206] In one embodiment, detection is by a labeled probe. Thetechniques involved are well known to those of skill in the art and canbe found in many standard books on molecular protocols. See Sambrook etal., 1989. For example, chromophore or radiolabel probes or primersidentify the target during or following amplification.

[0207] One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

[0208] In addition, the amplification products described above may besubjected to sequence analysis to identify specific kinds of variationsusing standard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optimal sequencing (Pignon et al, 1994). Thepresent invention provides methods by which any or all of these types ofanalyses may be used. Using the sequences disclosed herein,oligonucleotide primers may be designed to permit the amplification ofsequences throughout the STARS genes that may then be analyzed by directsequencing.

[0209] (vi) Kit Components

[0210] All the essential materials and reagents required for detectingand sequencing STARS and variants thereof may be assembled together in akit. This generally will comprise preselected primers and probes. Alsoincluded may be enzymes suitable for amplifying nucleic acids includingvarious polymerases (RT, Taq, Sequenase™ etc.), deoxynucleotides andbuffers to provide the necessary reaction mixture for amplification.Such kits also generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachprimer or probe.

[0211] B. Immunologic Diagnosis

[0212] Antibodies of the present invention can be used in characterizingthe STARS content of healthy and diseased tissues, through techniquessuch as ELISAs and Western blotting. This may provide a screen for thepresence or absence of cardiomyopathy or serve as a predictor of heartdisease.

[0213] The use of antibodies of the present invention in an ELISA assayis contemplated. For example, anti-STARS antibodies are immobilized ontoa selected surface, preferably a surface exhibiting a protein affinitysuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed material, it is desirable to bind or coatthe assay plate wells with a non-specific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of powdered milk. This allowsfor blocking of non-specific adsorption sites on the immobilizingsurface and thus reduces the background caused by non-specific bindingof antigen onto the surface.

[0214] After binding of antibody to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with the sampleto be tested in a manner conducive to immune complex (antigen/antibody)formation.

[0215] Following formation of specific immunocomplexes between the testsample and the bound antibody, and subsequent washing, the occurrenceand even amount of immunocomplex formation may be determined bysubjecting same to a second antibody having specificity for STARS thatdiffers from the first antibody. Appropriate conditions preferablyinclude diluting the sample with diluents such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/Tween®. These addedagents also tend to assist in the reduction of nonspecific background.The layered antisera is then allowed to incubate for from about 2 toabout 4 hr, at temperatures preferably on the order of about 25° C. toabout 27° C. Following incubation, the antisera-contacted surface iswashed so as to remove non-immunocomplexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween®, or boratebuffer.

[0216] To provide a detecting means, the second antibody will preferablyhave an associated enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody-boundsurface with a urease or peroxidase-conjugated anti-human IgG for aperiod of time and under conditions which favor the development ofimmunocomplex formation (e.g., incubation for 2 hr at room temperaturein a PBS-containing solution such as PBS/Tween®).

[0217] After incubation with the second enzyme-tagged antibody, andsubsequent to washing to remove unbound material, the amount of label isquantified by incubation with a chromogenic substrate such as urea andbromocresol purple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonicacid (ABTS) and H₂O₂, in the case of peroxidase as the enzyme label.Quantitation is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectrum spectrophotometer.

[0218] The preceding format may be altered by first binding the sampleto the assay plate. Then, primary antibody is incubated with the assayplate, followed by detecting of bound primary antibody using a labeledsecond antibody with specificity for the primary antibody.

[0219] The antibody compositions of the present invention will findgreat use in immunoblot or Western blot analysis. The antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. Immunologically-based detection methods for use inconjunction with Western blotting include enzymatically-, radiolabel-,or fluorescently-tagged secondary antibodies against the toxin moietyare considered to be of particular use in this regard.

[0220] C. Treating Defects in STARS Expression or Function

[0221] The present invention also involves, in another embodiment, thetreatment of disease states related to the aberrant expression and/orfunction of STARS. In particular, it is envisioned that STARS activityplays a role in development of cardiac tissue. Thus, increasing levelsof STARS, or compensating for mutations that reduce or eliminate theactivity of STARS, are believed to provide therapeutic intervention incertain cardiomyopathies.

[0222] In addition, by increasing levels of STARS, it is possible thatdefects in other cardiac genes may be compensated for. STARS may be ableto overcome deficiencies in the expression of other cardiac factors.

[0223] There also may be situations where one would want to inhibitSTARS function or activity, for example, where overexpression orunregulated expression had resulted in cardiac dysfunction. In thiscase, one would take steps to interfere with or block the expression ofSTARS, or inhibit its activity.

[0224] D. Genetic Based Therapies

[0225] One of the therapeutic embodiments contemplated by the presentinventors is intervention, at the molecular level, with the eventsinvolved in cardiac failure. Specifically, the present inventors intendto provide, to a cardiac cell, an expression construct capable ofproviding STARS to that cell. The lengthy discussion of expressionvectors and the genetic elements employed therein is incorporated intothis section by reference. Particularly preferred expression vectors areviral vectors such as adenovirus, adeno-associated virus, herpesvirus,vaccinia virus and retrovirus. Also preferred areliposomally-encapsulated expression vectors.

[0226] Those of skill in the art are aware of how to apply gene deliveryto in vivo situations. For viral vectors, one generally will prepare aviral vector stock. Depending on the kind of virus and the titerattainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹,1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the patient. Similarfigures may be extrapolated for liposomal or other non-viralformulations by comparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below. Variousroutes are contemplated, including local and systemic, but targetedprovision to the heart is preferred. (See, for example Hammond, et al.,supra, hereby incorporated by reference in its entirety.)

[0227] E. Combined Therapy

[0228] In many clinical situations, it is advisable to use a combinationof distinct therapies. Thus, it is envisioned that, in addition to thetherapies described above, one would also wish to provide to the patientmore “standard” pharmaceutical cardiac therapies. Examples of standardtherapies include so-called “beta blockers”, anti-hypertensives,cardiotonics, anti-thrombotics, vasodilators, hormone antagonists,endothelin antagonists, cytokine inhibitors/blockers, calcium channelblockers, phosphodiesterase inhibitors and angiotensin type 2antagonists. Also envisioned are combinations with pharmaceuticalsidentified according to the screening methods described herein.

[0229] Combinations may be achieved by contacting cardiac cells with asingle composition or pharmacological formulation that includes bothagents, or by contacting the cell with two distinct compositions orformulations, at the same time, wherein one composition includes theexpression construct and the other includes the agent. Alternatively,the STARS therapy may precede or follow the other agent treatment byintervals ranging from minutes to weeks. In embodiments where the otheragent and expression construct are applied separately to the cell, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent and expressionconstruct would still be able to exert an advantageously combined effecton the cell. In such instances, it is contemplated that one wouldcontact the cell with both modalities within about 12-24 hours of eachother and, more preferably, within about 6-12 hours of each other, witha delay time of only about 12 hours being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

[0230] It also is conceivable that more than one administration ofeither a STARS gene, protein or STARS agent, or the other agent will bedesired. Various combinations may be employed, where STARS is “A” andthe other agent is “B”, as exemplified below: A/B/A B/A/B B/B/A A/A/BB/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/AB/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

[0231] Other combinations are contemplated as well.

[0232] F. Formulations and Routes for Administration to Patients

[0233] Where clinical applications are contemplated, it will benecessary to prepare pharmaceutical compositions—expression vectors,virus stocks and drugs—in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

[0234] One will generally desire to employ appropriate salts and buffersto render delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

[0235] The active compositions of the present invention may includeclassic pharmaceutical preparations. Administration of thesecompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal, intravascular or intravenous injection.Such compositions would normally be administered as pharmaceuticallyacceptable compositions, described supra.

[0236] The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0237] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion, and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

[0238] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent witha variety of the other ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0239] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0240] For oral administration the polypeptides of the present inventionmay be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

[0241] The compositions of the present invention may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

[0242] Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. For example, one dosage could bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

[0243] V. Drug Formulations and Routes for Administration to Patients

[0244] Where clinical applications are contemplated, pharmaceuticalcompositions will be prepared—e.g., expression vectors, virus stocks anddrugs—in a form appropriate for the intended application. Generally,this will entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals.

[0245] One will generally desire to employ appropriate salts and buffersto render delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector or cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes solvents,buffers, solutions, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the likeacceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well know inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the vectors or cells of the compositions.

[0246] The active compositions of the present invention may includeclassic pharmaceutical preparations. Administration of thesecompositions according to the present invention may be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, as described supra.

[0247] The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

[0248] The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy syringability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

[0249] Sterile injectable solutions may be prepared by incorporating theactive compounds in an appropriate amount into a solvent along with anyother ingredients (for example as enumerated above) as desired, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient(s) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0250] For oral administration the polypeptides of the present inventiongenerally may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

[0251] The compositions of the present invention generally may beformulated in a neutral or salt form. Pharmaceutically-acceptable saltsinclude, for example, acid addition salts (formed with the free aminogroups of the protein) derived from inorganic acids (e.g., hydrochloricor phosphoric acids, or from organic acids (e.g., acetic, oxalic,tartaric, mandelic, and the like. Salts formed with the free carboxylgroups of the protein can also be derived from inorganic bases (e.g.,sodium, potassium, ammonium, calcium, or ferric hydroxides) or fromorganic bases (e.g., isopropylamine, trimethylamine, histidine, procaineand the like.

[0252] Upon formulation, solutions are preferably administered in amanner compatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

[0253] VI. Methods of Making Transgenic Mice

[0254] A particular embodiment of the present invention providestransgenic animals that contain a selectable or screenable markerprotein under the control of a STARS promoter. Transgenic animalsexpressing a STARS encoding nucleic acid under the control of aninducible or a constitutive promoter, recombinant cell lines derivedfrom such animals, and transgenic embryos may be useful in determiningthe exact role that STARS plays in the development and differentiationof cardiomyocytes. Furthermore, this transgenic animal may provide aninsight into heart development. The use of constitutively expressedSTARS encoding nucleic acid provides a model for over- or unregulatedexpression. Also, transgenic animals which are “knocked out” for STARS,in one or both alleles, are contemplated.

[0255] In a general aspect, a transgenic animal is produced by theintegration of a given transgene into the genome in a manner thatpermits the expression of the transgene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; which is incorporated herein by reference), Brinsteret al. (1985); which is incorporated herein by reference in itsentirety) and in Hogan et al. (1994).

[0256] Typically, a gene flanked by genomic sequences is transferred bymicroinjection into a fertilized egg. The microinjected eggs areimplanted into a host female, and the progeny are screened for theexpression of the transgene. Transgenic animals may be produced from thefertilized eggs from a number of animals including, but not limited toreptiles, amphibians, birds, mammals, and fish.

[0257] DNA clones for microinjection can be prepared by any means knownin the art. For example, DNA clones for microinjection can be cleavedwith enzymes appropriate for removing the bacterial plasmid sequences,and the DNA fragments electrophoresed on 1% agarose gels in TBE buffer,using standard techniques. The DNA bands are visualized by staining withethidium bromide, and the band containing the expression sequences isexcised. The excised band is then placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags,extracted with a 1:1 phenol:chloroform solution and precipitated by twovolumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer(0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on anElutip-D™ column. The column is first primed with 3 ml of high saltbuffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn three times to bind DNA to the column matrix. After one wash with3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt bufferand precipitated by two volumes of ethanol. DNA concentrations aremeasured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 31 g/ml in 5 mM Tris,pH 7.4 and 0.1 mM EDTA. Other methods for purification of DNA formicroinjection are described in Hogan et al. (1986), in Palmiter et al.(1982); and in Sambrook et al. (1989).

[0258] In an exemplary microinjection procedure, female mice six weeksof age are induced to superovulate with a 5 IU injection (0.1 cc, ip) ofpregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours laterby a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG;Sigma). Females are placed with males immediately after hCG injection.Twenty-one hours after hCG injection, the mated females are sacrificedby CO2 asphyxiation or cervical dislocation and embryos are recoveredfrom excised oviducts and placed in Dulbecco's phosphate buffered salinewith 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cellsare removed with hyaluronidase (1 mg/ml). Pronuclear embryos are thenwashed and placed in Earle's balanced salt solution containing 0.5% BSA(EBSS) in a 37.5° C. incubator with a humidified atmosphere at 5% CO₂,95% air until the time of injection. Embryos can be implanted at thetwo-cell stage.

[0259] Randomly cycling adult female mice are paired with vasectomizedmales. C57BL/6 or Swiss mice or other comparable strains can be used forthis purpose. Recipient females are mated at the same time as donorfemales. At the time of embryo transfer, the recipient females areanesthetized with an intraperitoneal injection of 0.015 ml of 2.5%avertin per gram of body weight. The oviducts are exposed by a singlemidline dorsal incision. An incision is then made through the body walldirectly over the oviduct. The ovarian bursa is then torn withwatchmakers forceps. Embryos to be transferred are placed in DPBS(Dulbecco's phosphate buffered saline) and in the tip of a transferpipet (about 10 to 12 embryos). The pipet tip is inserted into theinfundibulum and the embryos transferred. After the transfer, theincision is closed by two sutures.

[0260] VII. Screening Assays

[0261] The present invention also contemplates the screening ofcompounds for various abilities to interact with and/or affect STARSexpression or function. Particularly preferred compounds will be thoseuseful in inhibiting or promoting the actions of STARS in regulating thedevelopment and differentiation of cardiomyocytes. In the screeningassays of the present invention, the candidate substance may first bescreened for basic biochemical activity—e.g., binding to a targetmolecule—and then tested for its ability to inhibit modulate activity,at the cellular, tissue or whole animal level.

[0262] A. Modulators and Assay Formats

[0263] i) Assay Formats

[0264] The present invention provides methods of screening formodulators of STARS expression and actin binding activity. In oneembodiment, the present invention is directed to a method of:

[0265] (a) providing an active STARS preparation;

[0266] (b) contacting said STARS preparation with a candidate modulator;and

[0267] (c) measuring the actin binding activity of said STARSpreparation,

[0268] In yet another embodiment, the assay looks not at actin binding,but at STARS expression. Such methods would comprise, for example:

[0269] (a) providing a cell in which a STARS promoter directs theexpression of a polypeptide;

[0270] (b) contacting said cell with a candidate modulator; and

[0271] (c) measuring the effect of said candidate modulator on saidpolypeptide,

[0272] the polypeptide may be STARS, or it may be an indicator protein.

[0273] ii) Candidate Substances

[0274] As used herein, the term “candidate substance” refers to anymolecule that may potentially modulate STARS expression or function. Thecandidate substance may be a protein or fragment thereof, a smallmolecule inhibitor, or even a nucleic acid molecule. The term “candidatemodulator” may be used in place of “candidate substance”. It may proveto be the case that the most useful pharmacological compounds will becompounds that are structurally related to compounds which interactnaturally with STARS. Creating and examining the action of suchmolecules is known as “rational drug design,” and include makingpredictions relating to the structure of target molecules.

[0275] The goal of rational drug design is to produce structural analogsof biologically active polypeptides or target compounds. By creatingsuch analogs, it is possible to fashion drugs which are more active orstable than the natural molecules, which have different susceptibilityto alteration or which may affect the function of various othermolecules. In one approach, one would generate a three-dimensionalstructure for a molecule like STARS, and then design a molecule for itsability to interact with STARS. Alternatively, one could design apartially functional fragment of STARS (binding, but no activity),thereby creating a competitive inhibitor. This could be accomplished byx-ray crystallography, computer modeling or by a combination of bothapproaches.

[0276] It also is possible to use antibodies to ascertain the structureof a target compound or inhibitor. In principle, this approach yields apharmacore upon which subsequent drug design can be based. It ispossible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site ofanti-idiotype would be expected to be an analog of the original antigen.The anti-idiotype could then be used to identify and isolate peptidesfrom banks of chemically- or biologically-produced peptides. Selectedpeptides would then serve as the pharmacore. Anti-idiotypes may begenerated using the methods described herein for producing antibodies,using an antibody as the antigen.

[0277] On the other hand, one may simply acquire, from variouscommercial sources, small molecule libraries that are believed to meetthe basic criteria for useful drugs in an effort to “brute force” theidentification of useful compounds. Screening of such libraries,including combinatorially generated libraries (e.g., peptide libraries),is a rapid and efficient way to screen large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third and fourth generation compounds modeled of active, butotherwise undesirable compounds.

[0278] Candidate compounds may include fragments or parts ofnaturally-occurring compounds or may be found as active combinations ofknown compounds which are otherwise inactive. It is proposed thatcompounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived or synthesized from chemicalcompositions or man-made compounds. Thus, it is understood that thecandidate substance identified by the present invention may bepolypeptide, polynucleotide, small molecule inhibitors or any othercompounds that may be designed through rational drug design STARStingfrom known inhibitors of hypertrophic response.

[0279] Other suitable inhibitors include antisense molecules, ribozymes,and antibodies (including single chain antibodies).

[0280] It will, of course, be understood that the screening methods ofthe present invention are useful in themselves notwithstanding the factthat effective candidates may not be found. The invention providesmethods for screening for such candidates, not solely methods of findingthem.

[0281] B. In Vitro Assays

[0282] A quick, inexpensive and easy assay to run is a binding assay.Binding of a molecule to a target may, in and of itself, be inhibitory,due to steric, allosteric or charge-charge interactions. This can beperformed in solution or on a solid phase and can be utilized as a firstround screen to rapidly eliminate certain compounds before moving intomore sophisticated screening assays. In one embodiment of this kind, thescreening of compounds that bind to a STARS molecule or fragment thereofis provided

[0283] The target may be either free in solution, fixed to a support,expressed in or on the surface of a cell. Either the target or thecompound may be labeled, thereby permitting determining of binding. Inanother embodiment, the assay may measure the inhibition of binding of atarget to a natural or artificial substrate or binding partner (such asSTARS). Competitive binding assays can be performed in which one of theagents (STARS for example) is labeled. Usually, the target will be thelabeled species, decreasing the chance that the labeling will interferewith the binding moiety's function. One may measure the amount of freelabel versus bound label to determine binding or inhibition of binding.

[0284] A technique for high throughput screening of compounds isdescribed in WO 84/03564. Large numbers of small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with, for example,STARS, and then washed fron the support to remove non-specifically boundprotein. Bound polypeptide is detected by various methods.

[0285] Purified target, such as STARS, can be coated directly ontoplates for use in the aforementioned drug screening techniques. However,non-neutralizing antibodies to the polypeptide can be used to immobilizethe polypeptide to a solid phase. Also, fusion proteins containing areactive region (preferably a terminal region) may be used to link anactive region to a solid phase.

[0286] C. In Cyto Assays

[0287] Various cell lines that express STARS can be utilized forscreening of candidate substances. For example, cells containing theSTARS gene with an engineered indicator can be used to study variousfunctional attributes of candidate compounds. In such assays, thecompound would be formulated appropriately, given its biochemicalnature, and contacted with a target cell.

[0288] Depending on the assay, culture may be required. As discussedabove, the cells may then be examined by virtue of a number of differentphysiologic assays (growth, size, Ca⁺⁺ effects). Alternatively,molecular analysis may be performed in which the function of STARS andrelated pathways may be explored. This involves assays such as those forprotein expression, enzyme function, substrate utilization, mRNAexpression (including differential display of whole cell or polyA RNA)and others.

[0289] D. In Vivo Assays

[0290] The present invention particularly contemplates the use ofvarious animal models. Transgenic animals may be created with constructsthat permit STARS expression and activity to be controlled andmonitored. The generation of these animals has been described elsewherein this document.

[0291] Treatment of these animals with test compounds will involve theadministration of the compound, in an appropriate form, to the animal.Administration will be by any route the could be utilized for clinicalor non-clinical purposes, including but not limited to oral, nasal,buccal, or even topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply.

[0292] E. Production of Inhibitors

[0293] In an extension of any of the previously described screeningassays, the present invention also provide for methods of producinginhibitors. The methods comprising any of the preceding screening stepsfollowed by an additional step of “producing the candidate substanceidentified as a modulator of” the screened activity.

VII. EXAMPLES

[0294] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Materials and Methods

[0295] Differential cDNA cloning. For subtractive screening byrepresentational difference analysis (RDA), hearts and the region of theembryo dorsal to the heart, including the neural fold and the firstthree somites were dissected from E8.25 mouse embryos. Total RNA wasisolated from the heart and the dorsal embryonic region using TRIZOL(Gibco). mRNA was purified using a mRNA purification kit (AmershamPharmacia) and converted into double-strand cDNA by the Superscriptchoice system for cDNA synthesis (Gibco). For subtraction screeningbetween heart (Tester) and other embryonic parts (Driver), the RDAmethod described by Hubank and Schatz 1999 was used. Briefly, the cDNAfrom both tissues was digested with DpnII and ligated with an R-linker.(The annealed oligonucleotides were: R-Bgl-12: 5′-GATCTGCGGTGA-3′ (SEQID:NO. 10) and R-Bgl-24: 5′-AGCACTCTCCAGCCTCTCACCGCA-3′ (SEQ ID:NO. 11))Using R-Bgl-24 as primer, cDNA was amplified by PCR and digested withDpnII again. cDNA from the heart was purified by gel extraction andligated with a J-linker. (The annealed oligonucleotides were: J-Bgl-12:5′-GATCTGTTCATG-3′ (SEQ ID:NO. 12) and J-Bgl-24:5′-ACCGACGTCGACTATCCATGAACA-3′ (SEQ ID:NO. 13)). Tester ligated withJ-linker and Driver cDNAs (ratio 1:100) were hybridized at 67° C. for 20hrs. PCR reactions were then performed by primer J-Bgl-24 to yield a PCRproduct referred to as DPI (Differential product). DPI was digested withDpnII as a new tester and ligated with N-linker (The annealedoligonucleotides were: N-Bgl-12: 5′-GATCTTTCCATCG-3′ (SEQ ID:NO. 14) andN Bgl-24: 5′-AGGCAACTGTGCTATCCGAGGGAA-3′ (SEQ ID:NO. 15)).N-linker-ligated DPI was hybridized with Driver (ratio 1:800) andamplified by PCR two times using N-oligonucleotides as primer. Linkersfor tester were replaced with J-oligonucleotides in the third round, andN-oligonucleotides in the fourth round of hybridization. The final PCRproducts, referred to as DPIV, were cloned into the TA cloning vector(pGEMT easy, Promega) and inserted DPIV fragments were amplified by PCRusing N-Bgl-24 as primers to make two identical dot blots. One dot blotwas hybridized with tester probe, and the other was hybridized withdriver probe to confirm the differential expression of DPIV fragments.

[0296] To obtain a full-length STARS cDNA, an E10.5 mouse heart cDNAlibrary (Stratagene) was screened using a 342 bp cDNA fragment isolatedby RDA. The longest positive clone contained a 375-amino acid openreading frame, without a stop codon 5′ of the first methionine in thesequence. To obtain further 5′ sequence, adult mouse heart and skeletalmuscle cDNA libraries (Clontech) were screened with a 400 bp cDNAfragment from the 5′ end of the longest cDNA as a probe. 5′-RACE wasalso performed using the SMART 5′-RACE kit (Clontech) and the RLM firstchoice 5′-RACE kit (Ambion) following the manufacturer's instructions.5-RACE was performed with mRNA from human skeletal muscle (Clontech) andfrom mouse heart and skeletal muscle. There were no stop codons upstreamof the first methionine.

[0297] Northern Blot Analysis. Northern blot analysis was performedusing mouse and human multiple tissue Northern blots (Clontech) or totalRNA isolated from C2 skeletal muscle cells and mouse hearts by theTRIZOL reagent.

[0298] C2 skeletal muscle cells were maintained in DMEM with 20% FBS(growth medium) and were induced to differentiate by transfer to mediumwith 2% horse serum (differentiation medium). Transgenic mice harboringan α-MHC-calcineurin transgene have been described previously (Molkentinet al., 1998). ³²P-labeled probes were prepared from a full-length mouseSTARS cDNA and a partial human cDNA. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) transcripts were measured as a control.Hybridizations were performed in Quick Hyb (Clontech).

[0299] In situ Hybridization. RNA probes corresponding to the sense andthe anti-sense strands of the STARS cDNA were prepared. In situhybridization was performed on sagittal sections of mouse embryos, asdescribed (Benjamin et al., 1997).

[0300] STARS Expression Plasmids. STARS expression constructs weregenerated using the pcDNA3.1 mammalian expression vector (Invitrogen)which was modified to contain an amino-terminal Myc-tag. Constitutivelyactive RhoA (L63) and C3 transferase in Prk5 were generously provided byDr. Alan Hall (University College, London, United Kingdom). TheSM22-luciferase reporter 1343 bp promoter has been previously described(Li et al., 1995). The smooth muscle α actin-luciferase reportercontained the 4 kb promoter region ligated into the pBL3basic vector(Promega). The ANF reporter contained the 638 bp promoter region (agenerous gift from Dr. Mona Nemer, Universite de Montreal, Montreal,Quebec, Canada). The 4×cfos reporter contained four tandem copies of thecfos CArG box with flanking sequences, as described (Chang et al.,2001). CMV-lacZ (Promega) was used as an internal control fortransfection efficiency.

[0301] Transient Transfections, Reporter Assays and ImmunofluorescenceMicroscopy. COS cells were maintained in DMEM with 10% FBS. Eighteenhours after plating, the medium was changed to serum-free medium andtransfections were performed at 50% confluency using FuGENE reagent-6(Roche Molecular Biochemicals) following manufacturer's instructions.Each transfection used 0.15 μg of plasmid DNA for the reporter assays,and 0.5-1.0 μg immunohistochemistry. Cytochalasin D 2 μM(Sigma-Aldrich), and Y-27632 75 μM (Tocris) were added directly justafter transfection. Latrunculin B 0.5 μM (dissolved in 10% ethanol,Calbiochem) was added 12 hr after transfection and 10% ethanol (finalconcentration 0.01%) was added to control wells. Cells were harvested 36hr after transfection and their luciferase and β-galactosidase activitywere measured.

[0302] For measuring immunofluorescence, cells were rinsed with PBS andfixed in 4% paraformaldehyde for 10 min on ice. After rinsing with PBS,COS cells were incubated in 0.1% Triton X-100 in PBS for 5 min on ice.For rat neonatal cardiomyocytes, 1% Triton X-100 in PBS was used. Cellswere blocked for 20 min at room temperature in 0.5% BSA in PBS for COScells and in 1% BSA plus 3% goat serum in PBS for rat cardiomyocytes.Then, cells were incubated with first antibody diluted in 0.5% BSA inPBS for 30-60 min at room temperature or overnight at 4° C. Anti c-mycmonoclonal antibody 9E10 (Santa Cruz) was used at a 1:200 dilution.Other antibodies were anti α-actinin monoclonal antibody (1:400;Sigma-Aldrich), α-tubulin antibody (1:2000; Sigma-Aldrich) andanti-vimentin antibody (1:500; Sigma-Aldrich). Secondary antibodies wereanti mouse/rabbit IgG FITC or Texas red (Vector) used at a 1:200dilution incubated with or without Phalloidin-TRITC (1:500; SigmaAldrich).

[0303] Western Blot and Co-immunoprecipitation Assay.Immunoprecipitation and western blot analysis was performed as describedpreviously (Lu et al., 2000). For immunoprecipitation, cells wereincubated with 1 μg anti c-myc polyclonal antibody A14 (Santa Cruz).Western blots were performed with anti-actin monoclonal antibody C4(1:100; Roche Molecular Biochemicals) or anti c-myc monoclonal antibody9E10 (1:1000; Santa Cruse) followed by incubation with secondaryantibody HRP-conjugated anti mouse-IgG (Santa Cruse, Vector). To detectsignals, blots were incubated with ECL (Santa Cruz) for 1 min, andexposed to film (Eastman Kodak Co).

[0304] Adenoviral transduction and infection to cardiomyocytes. The fulllength STARS cDNA including a Kozak consensus sequence was ligated intopACCMViresAdeno (a kind gift from Dr. Robert Gerard) to generateadenovirus expressing STARS and GFP protein by Cre-lox recombination(Aoki et al., 1999). Neonatal rat cardiomyocytes were isolated asdescribed previously (Nicol et al., 2001). After 36 hr, cardiomyocyteswere infected for 2 hr with STARS- and GFP-expressing adenovirus at amultiplicity of infection of 100. After infection, cells were culturedin non-serum medium (DMEM:Medium199 4:1, penicillin/streptomycin).

[0305] GST-STARS purification and sedimentation assays. A cDNA encodingthe full-length STARS open reading frame was cloned in-frame into vectorpGEXKG (Guan and Dixon, 1991). BL21-DE3 cells containing the GST-fusionexpression plasmid were grown to an optical density of 0.5 and inducedwith 1 mM IPTG for 3 hr at 370 C. GST-STARS was purified by GST-affinitychromatography using standard techniques. Purified actin and actininwere purchased from Cytoskeleton, Inc. BSA was purchased fromSigma-Aldrich. Actin sedimentation assays were performed as recommendedby Cytoskeleton, Inc. Supernatants and pellets were analyzed by SDS-PAGEand coomassie blue staining.

[0306] Antibody production. To raise an anti-STARS antibody, the fulllength cDNA was cloned in frame with glutathione-S-transferase in pGEXKG(Guan and Dixon, 1991), which was tranformed into BL21 DE3 codon plus(Stratagene). Protein induction and purification was performed followingstandard protocol. Rabbit immunization was conducted by CocalicoBiologicals. For immunostaining and western blots, IgG was purified fromantisera with protein A sepharose beads (Zymed).

Example 2 Results

[0307] Evolutionary conservation of STARS proteins. A differential cDNAscreen was performed for novel genes expressed in the mouse heart tubeat E8.25, but not in other regions of the E8.25 embryo. Positive clonesrepresenting heart tube-specific genes were used for whole-mount in situhybridization to confirm their cardiac-specificity. One of thecardiac-specific cDNAs isolated in the screen encoded a novel 375-aminoacid protein that is referred to as striated muscle activator of RhoSignalin (STARS), because it is expressed specifically in striatedmuscle where it binds and bundles actin (see below). The predicted openreading frame of STARS did not contain recognizable protein motifs.Database searches for related genes revealed an apparent human orthologencoded by a gene located on chromosome 8q23. A partial cDNA encoding aSTARS-like protein was also identified in the zebrafish EST database(FIG. 1). No other related genes were identified in searches of themouse and human genome sequences, indicating that STARS is a single genewithout related family members. Genomic sequences with the potential toencode proteins with high homology to the carboxy-terminal 142 aminoacids of STARS were also identifed in C. elegans (F36F2.1, T04A8.4) andDrosophila melanogaster (FIG. 1).

[0308] STARS is expressed specifically in cardiac and skeletal muscle.Consistent with the cDNA subtraction scheme, STARS transcripts weredetected by in situ hybridization only in the primitive heart tube atE8.75 (FIG. 2A). Thereafter, STARS expression was maintained in theheart and was also detected in skeletal muscle after E10.5 (data notshown). Northern analysis of adult tissues revealed three STARStranscripts in mouse heart and skeletal muscle and two transcripts inthese human tissues (FIG. 2B). Sequencing of multiple independent cDNAsrevealed only a single open reading frame with no evidence foralternative splicing within the protein coding region. Therefore, it isbelieved that the multiple transcripts reflect the presence of different3′-untranslated sequences.

[0309] In the C2 skeletal muscle cell line, STARS expression wasundetectable in proliferating myoblasts, but was upregulated after threedays in differentiation medium, following the formation of myotubes(FIG. 2C). Thus, STARS appears to be a relatively late marker forskeletal muscle differentiation.

[0310] STARS was examined to determine if it was regulated duringhypertrophic growth of the heart, which is associated with up-regulationof a specific set of cardiac genes. Expression of activated calcineurinin the heart results in profound hypertrophy that ultimately progressesto dilated cardiomyopathy (Molkentin et al., 1998). STARS expression wasupregulated dramatically in calcineurin transgenic mice at 3 months ofage, when hearts showed extreme concentric hypertrophy without obviousleft ventricular failure (FIG. 2D). By 6 months of age, when hearts fromthese mice had dilated and were in late stages of failure, STARSexpression was further up-regulated. Cardiac expression of STARS wasalso up-regulated following banding of the thoracic aorta, which resultsin hypertrophy due to pressure-overload.

[0311] STARS localization in cardiomyocytes. Western blot analysis ofadult mouse heart extracts an anti-STARS antibody revealed a singleprotein species of 45 kD, similar to the predicted size of STARS (FIG.3A). This band comigrated with a Myc-tagged STARS protein expressed intransfected COS cells.

[0312] To determine the subcellular distribution of STARS, rat primarycardiomyocytes were immunostained with the STARS antibody (FIG. 3B,a&d). STARS staining showed a periodicity reminiscent of sarcomericlocalization. The sarcomeric localization of STARS abuts the Z-line onboth sides as demonstrated by a partial overlap with α-actinin (FIG. 3B,b&c, e&f). This periodicity demonstrates that STARS is localized to theI-band of the sarcomere. In addition to I-band localization, a portionof STARS localizes to sarcomeric structures between Z-lines (FIG. 3B,c&f). A schematic of STARS localization is depicted in FIG. 3C.Together, these results indicated that STARS functions as amuscle-specific actin-binding protein.

[0313] Mapping the F-actin binding domain of STARS. To furtherinvestigate the function of STARS, the subcellular localization of aMyc-tagged STARS protein in transiently transfected COS cells wasexamined. STARS strongly colocalized with F-actin in stress fibers andmembrane ruffles, which were also stained by Phalloidin-TRITC (FIG. 4A;c, d and e). STARS-transfected cells also exhibited an increased numberof actin stress fibers and especially prominent thick fibers, indicatingthat ectopic STARS expression induced stress fiber formation andbundling (FIG. 4A; f, g and h). Other structural proteins, includingtubulin, vimentin, and keratin did not colocalize with STARS (data notshown).

[0314] To map the region of STARS that mediated the association withF-actin, immunofluorescence was performed with a series of STARSdeletion mutants in transfected COS cells. Deletion mutants lackingresidues from the carboxy-terminus into the conserved region of STARS(mutants N346, N323, N304, N279, N233, N100, and N101-233), and adeletion mutant within the conserved region (C96), did not affect theassociation with F-actin, but abolished the ability of STARS to bundleactin stress fibers (FIG. 4A; l, m, n and 5, and data not shown). Incontrast to C96, a deletion mutant that encoded only the conservedregion (C 142) not only associated with F-actin, but also increasedstress fibers, although bundled stress fibers were not as frequent asseen with the wild-type protein (FIG. 4A; i, j and, k). Deletion mutantDel234-279 was able to associate with F-actin, but lost the ability tobundle actin fibers. Smaller sub-deletions of this region retained theability to bundle actin, indicating that the amino acid residuesresponsible for this function were distributed across residues 234-279.These findings indicated that the conserved carboxy-terminal domain ofSTARS was responsible for actin-bundling and that there were multiple,non-overlapping regions of the protein capable of weakly associatingwith F-actin in vivo.

[0315] Co-immunoprecipitation of STARS with actin. The actin bindingfunction of STARS was further investigated by co-immunoprecipitationexperiments in COS cells transiently transfected with Myc-taggedproteins. As shown in FIG. 4B, the wild-type protein and the conservedcarboxy terminal region (C142) co-immunoprecipitated with actin.Deletions that removed the carboxy-terminal portion of the conservedregion did not bind actin (mutants N346, N323, N304, N279, N233, N100,and N101-233), whereas small internal deletion mutants within theconserved region (Del 234-247, Del 248-262, Del 263-279) retained weakactin-binding activity in this assay. The larger deletion mutant in thisregion (Del 234-279) failed to co-immunoprecipitate with actin. Theseresults revealed a direct correlation between actin binding/bundlingactivity in vivo and co-immunoprecipitation of STARS with actin (FIG.5).

[0316] STARS stimulates SRF-dependent transcription via actinpolymerization. Recent studies have shown that stabilization of theactin cytoskeleton stimulates the transcriptional activity of SRF (Macket al., 2001), but the proteins involved in this phenomenon have notbeen identified. To examine whether STARS might participate in asignaling pathway between the cytoskeleton and the nucleus, the promoterof the SM22 gene, which is regulated by SRF in muscle cells (Li et al.,1997; Kim et al., 1997), was tested to determine if it was responsive toSTARS. Remarkably, STARS stimulated the expression of an SM22 luciferasereporter by 40-fold in transiently transfected COS cells (FIG. 6A).Mutation of the CArG boxes in the SM22 promoter abolished responsivenessto STARS, demonstrating the involvement of SRF in this response. In F9cells, which have low endogenous SRF expression (Li et al., 1997), STARShad a minimal effect on the SM22 promoter (FIG. 6A), further indicatingthat the effects of STARS were mediated by SRF. Stimulation of SRFactivity is not a general property of actin-binding proteins, asα-actinin has no effect on SRF activity (data not shown). STARS alsoweakly stimulated the α-smooth muscle actin promoter, which is regulatedby SRF (Mack and Owens, 1999), and it activated an E1b promoter linkedto four tandem copies of the CArG box and flanking sequences from thec-fos promoter (FIG. 6B). However, it did not stimulate the atrialnatriureticfactor (ANF) promoter (FIG. 6B) which is also regulated bySRF (Hines et al., 1999). It also did not activate the cytomegaloviruspromoter (data not shown).

[0317] A summary of the properties of STARS deletion mutants is shown inFIG. 5. Comparison of the complete set of STARS deletion mutants showeda direct correlation between the ability to bind actin and bundle actinfibers and to stimulate SM22 transcription (FIG. 6C). The conservedcarboxy terminal region of STARS was necessary and sufficient tostimulate SRF activity, as demonstrated by the finding that mutant C142was as effective as the full-length protein in activatingSM22-luciferase.

[0318] Effects of actin depolymerizating agents on SM22 promoteractivation by STARS. To examine whether STARS stimulates SRF activityvia its effects on actin dynamics, COS cells were treated withcytochalasin D (CD), which prevents actin polymerization (Sampath andPollard, 1991), and latrunculin B (LB), which sequesters monomeric actin(Morton et al., 2000). In STARS-transfected cells, only short fragmentsof disrupted stress fibers were observed in the presence of 2 μM CD or0.5 μM LB (data not shown). CD itself increased SM22 promoter activityby 8-fold, consistent with previoius results (Sotiropoulos et al., 1999;Mack et al., 2001). STARS increased promoter activity 18-fold in thepresence of CD. In contrast, LB treatment abolished SM22-luciferaseactivity even in cells transfected with STARS (FIG. 6D). These findingsindicated that STARS might stimulate SRF activity by reducing thecellular pool of G-actin as a result of its ability to enhance actinpolymerization. The opposing effects of CD and LB are likely to reflecttheir differential effects on the pool of G-actin; whereas CD dimerizesG-actin (Goddette and Frieden, 1986), LB sequesters G-actin (Morton etal., 2000).

[0319] Involvement of RhoA in SRF activation by STARS. In light of theability of RhoA to stimulate SRF activity by promoting actinpolymerization (Mack et al., 2001; Sotiropoulos et al., 1999), theeffects of a constitutively active RhoA mutant (L63) and STARS on SM22promoter activity were compared. As shown in FIG. 6E, STARS and RhoA L63activated the SM22 promoter to comparable levels and together stimulatedactivity to higher levels.

[0320] Rho signaling is inhibited by the Rho kinase inhibitor Y-27632,which inhibits stress fiber formation (Maekawa et al., 1999), and C3transferase, which specifically ADP-ribosylates RhoA (Nemoto et al.,1992). Treatment of COS cells with Y-27632 (75 μM) or transfection witha C3 expression plasmid reduced the stimulatory activity of STARS on theSM22 promoter by 55% and 88%, respectively (FIG. 6F). Together, theseresults indicate that STARS requires a basal level of RhoA activity toenhance SRF activity.

Example 3 Discussion

[0321] Stimulation of SRF-dependent transcription by STARS. Previousstudies indicated that G-actin suppresses the activity of SRF(Sotiropoulos et al., 1999), and several observations indicate thatSTARS activates SRF by relieving this repressive influence. For example,agents such as latrunculin B, which sequesters G-actin monomers (Mortonet al., 2000), inhibit the activity of STARS. The ability of STARS tostimulate actin polymerization and cross-linking would also be expectedto reduce the G-actin pool. It is important to point out that thestimulation of actin polymerization per se is not sufficient to accountfor the effects of STARS on SRF activity, because STARS is able toincrease SRF activity in the presence of cytochalasin D, which preventsactin polymerization and also stimulates SRF (Sotiropoulos et al., 1999;Mack et al., 2001).

[0322] A working model of a potential mechanism of action of STARSwithin the context of the regulation of actin dynamics is shown in FIG.7. Actin treadmilling determines the relative distribution of themonomeric G-actin and polymerized F-actin states. Previous studiessuggested that G-actin suppresses the activity of SRF (Sotiropoulos etal., 1999). The results suggest that STARS activates SRF by relievingthis repressive influence, as a result of its ability to stimulate actinpolymerization and thereby reduce the G-actin pool. Such a mechanismwould account for the ability of latrunculin B, which sequesters G-actinmonomers (Morton et al., 2000), to interfere with the activity of STARS.

[0323] While polymerization of actin by STARS appears to be coupled toSRF activation, stimulation of actin polymerization per se is notsufficient to account for the effects of STARS on SRF activity, becauseSTARS is able to increase SRF activity in the presence of cytochalasinD, which prevents actin polymerization by binding to the plus-end ofF-actin, where it blocks further addition of actin subunits.Cytochalasin D also stimulates SRF activity alone, presumably byreducing the level of free G-actin monomers (Sotiropoulos et al., 1999;Mack et al., 2001).

[0324] In principle, G-actin could inhibit SRF directly or it couldsequester cofactors required for SRF activation (Sotiropoulos et al.,1999). In light of the actin-binding properties of STARS, it maystimulate SRF by acting as a sink to sequester G-actin and therebyrelieve repression on SRF. G-actin has been shown to shuttle to thenucleus (Wada et al., 1998) and also to be contained within the SWI-SNFchromatin remodeling complex (Van Etten et al., 1994; Zhao et al., 1998;Rando et al., 2000). Whether STARS might alter the incorporation ofactin into this complex is a possiblity. Since STARS is localized to thecytoplasm, it may not stimulate transcription by associating directlywith SRF or other transcriptional components.

[0325] SRF activates muscle-specific transcription by recruitingmyogenic transcription factors, such as GATA4, Nkx2.5 and myocardin(Chen and Schwartz, 1996; Belaguli et al., 2000; Morin et al., 2001;Wang et al., 2001). Although STARS is a muscle-specific protein, itsability to stimulate SRF activity in nonmuscle cells indicates that itdoes not require these myogenic transcription factors for activity.STARS is upregulated after the onset of myocyte differentiation and mayact as an actin cross-linker during myofibrillogenesis, thereby furtherenhancing SRF activity in differentiated muscle cells and reinforcingthe expression of SRF-dependent sarcomeric genes.

[0326] Regulation of STARS activity, SRF activity and muscle geneexpression by Rho signaling. Rho signaling promotes the formation ofF-actin and could deplete the G-actin pool. Sotiropoulus concluded thatthe effects of Rho on SRF are secondary to its effects on thecytoskeleton and are mediated by a decrease in G-actin as a result ofactin polymerization (Sotiropoulos et al., 1999). Stimulation of SRFactivity by STARS appears to require at least a basal level of RhoAsignaling, as demonstrated by the ability of the Rho kinase inhibitorY-27632 and C3 transferase to diminish SRF activation by STARS.Conversely, constitutively active RhoA enhances the stimulatory effectof STARS on SRF activity. The fact that Y-27632 only partially inhibitsSTARS activity may indicate the involvement of other Rho effectors inthe mechanism for STARS action. It is also possible that theseinhibitors interfere with the SRF-activating properties of STARS throughan indirect mechanism due to changes in cell morphology.

[0327] Rho signaling plays an important role in muscle gene expression.Previous studies have shown that RhoA activates the skeletal muscleα-actin promoter, which is SRF-dependent (Wei et al., 1998; Wei et al.,2001). Activation of RhoA by Gq-coupled receptor agonists also inducespremyofibrils, myofibril organization and ANF expression in primarycardiomyocytes (Thorbum et al., 1997; Aoki et al., 1998; Hoshijima etal., 1998). STARS could not activate the ANF promoter, although RhoA canupregulate ANF expression in cardiomyocytes. This indicates that STARSdoes not activate Rho directly. Of course, stimulation of SRF activityby Rho does not require STARS, since Rho can activate SRF in nonmusclecells in which STARS is not expressed. In contrast, STARS requires basalRho activity to activate SRF, because the Rho inhibitor C3 and the Rhokinase inhibitor Y27632 decreased SM22 promoter activity significantly,indicating that STARS might have cross-talk or interaction with someeffector molecules of Rho and to Rho kinase. Taken together, the resultsindicate that stimulation of SRF activity by STARS activation is notmediated by the same effector-molecules that mediate ANF promoteractivation by RhoA in cardiomyocytes.

[0328] Linking the cytoskeleton and the sarcomere to muscle geneexpression. STARS expression is maintained in adult cardiac and skeletalmuscle and is dramatically up-regulated during hypertrophic growth ofthe heart in response to calcineurin activation and pressure-overload,which is associated with calcineurin activation (Leinwand, 2001).Sarcomere organization is a hallmark of cardiac hypertrophy. Duringhypertrophic growth of cardiomyocytes, STARS might organize newmyofibrils, whereas extreme overexpression might result indisorganization of actin bundles with resulting cardiac dysfunction asobserved in failing hearts.

[0329] The integrity of the cytoskeleton and sarcomere has a profoundinfluence on gene expression and growth of muscle cells. The actinbundling/binding and SRF-activating properties of STARS provide apotential link between myocyte structure and the program for muscle geneexpression.

[0330] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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1 15 1 1146 DNA Homo sapiens CDS (1)..(1146) 1 atg gct ccg ggc gaa aaggaa agc ggg gag ggc cca gcc aag agc gcc 48 Met Ala Pro Gly Glu Lys GluSer Gly Glu Gly Pro Ala Lys Ser Ala 1 5 10 15 ctc cgg aag ata cgc acagcc acc ctg gtc atc agc ttg gcc cga ggt 96 Leu Arg Lys Ile Arg Thr AlaThr Leu Val Ile Ser Leu Ala Arg Gly 20 25 30 tgg cag cag tgg gcg aat gagaac agc atc agg cag gcc cag gag cct 144 Trp Gln Gln Trp Ala Asn Glu AsnSer Ile Arg Gln Ala Gln Glu Pro 35 40 45 aca ggc tgg ctg ccg gga ggg acccag gac tca cct caa gct cct aaa 192 Thr Gly Trp Leu Pro Gly Gly Thr GlnAsp Ser Pro Gln Ala Pro Lys 50 55 60 cca atc aca ccc cct act tca cac cagaaa gct cag agt gcc cca aag 240 Pro Ile Thr Pro Pro Thr Ser His Gln LysAla Gln Ser Ala Pro Lys 65 70 75 80 tcg cca ccc cgc ctg cca gaa gga catgga gat gga caa agc tca gag 288 Ser Pro Pro Arg Leu Pro Glu Gly His GlyAsp Gly Gln Ser Ser Glu 85 90 95 aaa gcc cct gag gtt tct cac atc aaa aagaaa gag gtg tcc aaa acg 336 Lys Ala Pro Glu Val Ser His Ile Lys Lys LysGlu Val Ser Lys Thr 100 105 110 gtg gtc agc aag act tac gag aga gga ggggac gtg agc cac ctc agc 384 Val Val Ser Lys Thr Tyr Glu Arg Gly Gly AspVal Ser His Leu Ser 115 120 125 cac agg tac gag agg gat gct ggt gtg cttgaa cct ggg cag cca gag 432 His Arg Tyr Glu Arg Asp Ala Gly Val Leu GluPro Gly Gln Pro Glu 130 135 140 aat gac att gac aga atc ctc cac agc cacggc tcc cca acg cgg agg 480 Asn Asp Ile Asp Arg Ile Leu His Ser His GlySer Pro Thr Arg Arg 145 150 155 160 aga aaa tgt gcc aac ctg gtg tct gagcta acc aag ggc tgg aga gtg 528 Arg Lys Cys Ala Asn Leu Val Ser Glu LeuThr Lys Gly Trp Arg Val 165 170 175 atg gag cag gag gag ccc aca tgg aggagt gac agc gta gac aca gag 576 Met Glu Gln Glu Glu Pro Thr Trp Arg SerAsp Ser Val Asp Thr Glu 180 185 190 gac agc ggc tat gga gga gag gct gaggag agg ccc gag cag gat gga 624 Asp Ser Gly Tyr Gly Gly Glu Ala Glu GluArg Pro Glu Gln Asp Gly 195 200 205 gtg cag gtg gct gtg gtc agg atc aagcgc ccc ttg ccc tcc cag gta 672 Val Gln Val Ala Val Val Arg Ile Lys ArgPro Leu Pro Ser Gln Val 210 215 220 aac aga ttt aca gag aaa ctc aac tgcaaa gcc caa cag aaa tat agc 720 Asn Arg Phe Thr Glu Lys Leu Asn Cys LysAla Gln Gln Lys Tyr Ser 225 230 235 240 cca gtg ggc aac ttg aaa ggg agatgg cag cag tgg gct gat gaa cac 768 Pro Val Gly Asn Leu Lys Gly Arg TrpGln Gln Trp Ala Asp Glu His 245 250 255 ata caa tcc cag aag ctc aat cctttc agt gaa gag ttt gat tac gag 816 Ile Gln Ser Gln Lys Leu Asn Pro PheSer Glu Glu Phe Asp Tyr Glu 260 265 270 ctg gcc atg tcc acc cgc cta cacaaa gga gat gag ggc tat ggc cgc 864 Leu Ala Met Ser Thr Arg Leu His LysGly Asp Glu Gly Tyr Gly Arg 275 280 285 ccc aaa gaa gga acc aaa act gctgaa agg gcc aag cgt gct gag gag 912 Pro Lys Glu Gly Thr Lys Thr Ala GluArg Ala Lys Arg Ala Glu Glu 290 295 300 cac atc tac agg gaa atg atg gacatg tgc ttc att atc tgc aca atg 960 His Ile Tyr Arg Glu Met Met Asp MetCys Phe Ile Ile Cys Thr Met 305 310 315 320 gct cgc cac aga cga gat ggcaag atc cag gtt act ttt gga gat ctc 1008 Ala Arg His Arg Arg Asp Gly LysIle Gln Val Thr Phe Gly Asp Leu 325 330 335 ttt gac aga tac gtt cgt atttca gat aaa gta gtg ggc att ctc atg 1056 Phe Asp Arg Tyr Val Arg Ile SerAsp Lys Val Val Gly Ile Leu Met 340 345 350 cgt gcc agg aaa cat gga ctggta gac ttt gaa gga gag atg cta tgg 1104 Arg Ala Arg Lys His Gly Leu ValAsp Phe Glu Gly Glu Met Leu Trp 355 360 365 caa ggc cga gat gac cat gttgtg att acg cta ctc aag tga 1146 Gln Gly Arg Asp Asp His Val Val Ile ThrLeu Leu Lys 370 375 380 2 381 PRT Homo sapiens 2 Met Ala Pro Gly Glu LysGlu Ser Gly Glu Gly Pro Ala Lys Ser Ala 1 5 10 15 Leu Arg Lys Ile ArgThr Ala Thr Leu Val Ile Ser Leu Ala Arg Gly 20 25 30 Trp Gln Gln Trp AlaAsn Glu Asn Ser Ile Arg Gln Ala Gln Glu Pro 35 40 45 Thr Gly Trp Leu ProGly Gly Thr Gln Asp Ser Pro Gln Ala Pro Lys 50 55 60 Pro Ile Thr Pro ProThr Ser His Gln Lys Ala Gln Ser Ala Pro Lys 65 70 75 80 Ser Pro Pro ArgLeu Pro Glu Gly His Gly Asp Gly Gln Ser Ser Glu 85 90 95 Lys Ala Pro GluVal Ser His Ile Lys Lys Lys Glu Val Ser Lys Thr 100 105 110 Val Val SerLys Thr Tyr Glu Arg Gly Gly Asp Val Ser His Leu Ser 115 120 125 His ArgTyr Glu Arg Asp Ala Gly Val Leu Glu Pro Gly Gln Pro Glu 130 135 140 AsnAsp Ile Asp Arg Ile Leu His Ser His Gly Ser Pro Thr Arg Arg 145 150 155160 Arg Lys Cys Ala Asn Leu Val Ser Glu Leu Thr Lys Gly Trp Arg Val 165170 175 Met Glu Gln Glu Glu Pro Thr Trp Arg Ser Asp Ser Val Asp Thr Glu180 185 190 Asp Ser Gly Tyr Gly Gly Glu Ala Glu Glu Arg Pro Glu Gln AspGly 195 200 205 Val Gln Val Ala Val Val Arg Ile Lys Arg Pro Leu Pro SerGln Val 210 215 220 Asn Arg Phe Thr Glu Lys Leu Asn Cys Lys Ala Gln GlnLys Tyr Ser 225 230 235 240 Pro Val Gly Asn Leu Lys Gly Arg Trp Gln GlnTrp Ala Asp Glu His 245 250 255 Ile Gln Ser Gln Lys Leu Asn Pro Phe SerGlu Glu Phe Asp Tyr Glu 260 265 270 Leu Ala Met Ser Thr Arg Leu His LysGly Asp Glu Gly Tyr Gly Arg 275 280 285 Pro Lys Glu Gly Thr Lys Thr AlaGlu Arg Ala Lys Arg Ala Glu Glu 290 295 300 His Ile Tyr Arg Glu Met MetAsp Met Cys Phe Ile Ile Cys Thr Met 305 310 315 320 Ala Arg His Arg ArgAsp Gly Lys Ile Gln Val Thr Phe Gly Asp Leu 325 330 335 Phe Asp Arg TyrVal Arg Ile Ser Asp Lys Val Val Gly Ile Leu Met 340 345 350 Arg Ala ArgLys His Gly Leu Val Asp Phe Glu Gly Glu Met Leu Trp 355 360 365 Gln GlyArg Asp Asp His Val Val Ile Thr Leu Leu Lys 370 375 380 3 1128 DNA Musmusculus CDS (1)..(1128) 3 atg gct cca gga gaa agg gaa agg gag gcg gggccg gcc aag agt gcc 48 Met Ala Pro Gly Glu Arg Glu Arg Glu Ala Gly ProAla Lys Ser Ala 1 5 10 15 ctc cgg aag gtc cgc aca gca acc ctg gtt atcaat ttg gcc cga ggt 96 Leu Arg Lys Val Arg Thr Ala Thr Leu Val Ile AsnLeu Ala Arg Gly 20 25 30 tgg cag cag tgg gcg aat gag aac agt acc aaa caggcc cag gag cct 144 Trp Gln Gln Trp Ala Asn Glu Asn Ser Thr Lys Gln AlaGln Glu Pro 35 40 45 gca ggc tgg ctg ccg gga gca act cat gac gta cct aacgct cct aaa 192 Ala Gly Trp Leu Pro Gly Ala Thr His Asp Val Pro Asn AlaPro Lys 50 55 60 gaa gcc ggt cct tac cag cat gcc ccc aaa act ctg tct ccaaag cca 240 Glu Ala Gly Pro Tyr Gln His Ala Pro Lys Thr Leu Ser Pro LysPro 65 70 75 80 gat cga gac gga gag gga caa cac tca gaa gaa gcc acc gaggtc tcc 288 Asp Arg Asp Gly Glu Gly Gln His Ser Glu Glu Ala Thr Glu ValSer 85 90 95 cac att aaa agg aaa gag gtg acc aga acg gtt gtc agc aag gcttat 336 His Ile Lys Arg Lys Glu Val Thr Arg Thr Val Val Ser Lys Ala Tyr100 105 110 gag agg gga gga gat gtg aac tac ctg agc cac agg tat gag aatgat 384 Glu Arg Gly Gly Asp Val Asn Tyr Leu Ser His Arg Tyr Glu Asn Asp115 120 125 ggt ggc gtg tct gaa gct att cag cca gag aat gac att gac agaatc 432 Gly Gly Val Ser Glu Ala Ile Gln Pro Glu Asn Asp Ile Asp Arg Ile130 135 140 ctt ctt agt cac gac tcg cca aca cgg aga aga aaa tgc acc aacctg 480 Leu Leu Ser His Asp Ser Pro Thr Arg Arg Arg Lys Cys Thr Asn Leu145 150 155 160 gtg tct gag ctg acc aaa ggc tgg aaa gtg atg gaa cag gaagag ccc 528 Val Ser Glu Leu Thr Lys Gly Trp Lys Val Met Glu Gln Glu GluPro 165 170 175 acg tgg aag agt gac agc gta gac aca gag gac agt ggc tacgga ggg 576 Thr Trp Lys Ser Asp Ser Val Asp Thr Glu Asp Ser Gly Tyr GlyGly 180 185 190 gat atg gag gag agg cct gag caa gat gca gcg cct gtg gctcct gcc 624 Asp Met Glu Glu Arg Pro Glu Gln Asp Ala Ala Pro Val Ala ProAla 195 200 205 agg atc aaa cgc ccc ttg cac tcc cag gca aac agg tac tctgag cca 672 Arg Ile Lys Arg Pro Leu His Ser Gln Ala Asn Arg Tyr Ser GluPro 210 215 220 ctc aac tgt aag gcc cat cgg aaa tac agc caa gtg gac aacttg aaa 720 Leu Asn Cys Lys Ala His Arg Lys Tyr Ser Gln Val Asp Asn LeuLys 225 230 235 240 ggg agg tgg cag cag tgg gcc gat gaa cac gtc cag tcccag aag ctc 768 Gly Arg Trp Gln Gln Trp Ala Asp Glu His Val Gln Ser GlnLys Leu 245 250 255 aat ccc ttc agt gac gaa ttt gac tat gac cta gcc atgtcc act cgg 816 Asn Pro Phe Ser Asp Glu Phe Asp Tyr Asp Leu Ala Met SerThr Arg 260 265 270 ctc cac aag gga gac gag ggc tat ggc cgc ccc aaa gaggga agc aag 864 Leu His Lys Gly Asp Glu Gly Tyr Gly Arg Pro Lys Glu GlySer Lys 275 280 285 aca gct gaa agg gcc aag cga gcg gaa gag cac atc tatcgg gaa att 912 Thr Ala Glu Arg Ala Lys Arg Ala Glu Glu His Ile Tyr ArgGlu Ile 290 295 300 atg gaa ctg tgc ttt gtt atc cgc aca atg gct cgc cacaga cga gat 960 Met Glu Leu Cys Phe Val Ile Arg Thr Met Ala Arg His ArgArg Asp 305 310 315 320 ggc aag atc cag gtt act ttc gga gaa ctc ttt gatcgc tat gtt cgc 1008 Gly Lys Ile Gln Val Thr Phe Gly Glu Leu Phe Asp ArgTyr Val Arg 325 330 335 att tct gat aaa gtc gtg ggc atc ctc atg cgt gccagg aaa cac gga 1056 Ile Ser Asp Lys Val Val Gly Ile Leu Met Arg Ala ArgLys His Gly 340 345 350 ctg gtg cac ttt gaa gga gag atg cta tgg caa ggccga gac gac cat 1104 Leu Val His Phe Glu Gly Glu Met Leu Trp Gln Gly ArgAsp Asp His 355 360 365 gtt gtg att act ctc gtt gag taa 1128 Val Val IleThr Leu Val Glu 370 375 4 375 PRT Mus musculus 4 Met Ala Pro Gly Glu ArgGlu Arg Glu Ala Gly Pro Ala Lys Ser Ala 1 5 10 15 Leu Arg Lys Val ArgThr Ala Thr Leu Val Ile Asn Leu Ala Arg Gly 20 25 30 Trp Gln Gln Trp AlaAsn Glu Asn Ser Thr Lys Gln Ala Gln Glu Pro 35 40 45 Ala Gly Trp Leu ProGly Ala Thr His Asp Val Pro Asn Ala Pro Lys 50 55 60 Glu Ala Gly Pro TyrGln His Ala Pro Lys Thr Leu Ser Pro Lys Pro 65 70 75 80 Asp Arg Asp GlyGlu Gly Gln His Ser Glu Glu Ala Thr Glu Val Ser 85 90 95 His Ile Lys ArgLys Glu Val Thr Arg Thr Val Val Ser Lys Ala Tyr 100 105 110 Glu Arg GlyGly Asp Val Asn Tyr Leu Ser His Arg Tyr Glu Asn Asp 115 120 125 Gly GlyVal Ser Glu Ala Ile Gln Pro Glu Asn Asp Ile Asp Arg Ile 130 135 140 LeuLeu Ser His Asp Ser Pro Thr Arg Arg Arg Lys Cys Thr Asn Leu 145 150 155160 Val Ser Glu Leu Thr Lys Gly Trp Lys Val Met Glu Gln Glu Glu Pro 165170 175 Thr Trp Lys Ser Asp Ser Val Asp Thr Glu Asp Ser Gly Tyr Gly Gly180 185 190 Asp Met Glu Glu Arg Pro Glu Gln Asp Ala Ala Pro Val Ala ProAla 195 200 205 Arg Ile Lys Arg Pro Leu His Ser Gln Ala Asn Arg Tyr SerGlu Pro 210 215 220 Leu Asn Cys Lys Ala His Arg Lys Tyr Ser Gln Val AspAsn Leu Lys 225 230 235 240 Gly Arg Trp Gln Gln Trp Ala Asp Glu His ValGln Ser Gln Lys Leu 245 250 255 Asn Pro Phe Ser Asp Glu Phe Asp Tyr AspLeu Ala Met Ser Thr Arg 260 265 270 Leu His Lys Gly Asp Glu Gly Tyr GlyArg Pro Lys Glu Gly Ser Lys 275 280 285 Thr Ala Glu Arg Ala Lys Arg AlaGlu Glu His Ile Tyr Arg Glu Ile 290 295 300 Met Glu Leu Cys Phe Val IleArg Thr Met Ala Arg His Arg Arg Asp 305 310 315 320 Gly Lys Ile Gln ValThr Phe Gly Glu Leu Phe Asp Arg Tyr Val Arg 325 330 335 Ile Ser Asp LysVal Val Gly Ile Leu Met Arg Ala Arg Lys His Gly 340 345 350 Leu Val HisPhe Glu Gly Glu Met Leu Trp Gln Gly Arg Asp Asp His 355 360 365 Val ValIle Thr Leu Val Glu 370 375 5 612 DNA Zebra Fish modified_base (497) n =a, c, g or t/u 5 attaagacgg gaatcgtgac taaagctatt acgccgaagt gtaacgagtttggaaaggat 60 ttggtgagcg tgattaagga gaagatcaac accaatcaac tgacgactgaagacaccaaa 120 aatttcctag gaaatgaatc tcctactagg agacgctact gtggggggaaagcagggact 180 tttgttaaag caatcggacg gaaagaggga aagtcgatgg gatcgcgaagtagcagtttg 240 gatgctgatg acagcggtct tggggaggaa gcatctctga gcgacaacagcgatctgaac 300 gagaacgaac ccaagaaaca tgtcaacaga cacaagatta aagtgacaacgatgggtgac 360 ctgcggagcc gctggcagcg tttcgctgaa gatcacatgg agggccagaagctcaaccct 420 ttcagtgaag agtttgacta tgatcatgca atggccactc gactccacaaaggcgacgcg 480 ggctacggac gacccanaga aggatccaaa acagctcagc gagcagatcgagcccaaaag 540 cacatctacc gcgagatgga ggagatgtgc ttcatcatac gagacatgggccagcaggac 600 aaacagggcc aa 612 6 203 PRT Zebra Fish 6 Ile Lys Thr GlyIle Val Thr Lys Ala Ile Thr Pro Lys Cys Asn Glu 1 5 10 15 Phe Gly LysAsp Leu Val Ser Val Ile Lys Glu Lys Ile Asn Thr Asn 20 25 30 Gln Leu ThrThr Glu Asp Thr Lys Asn Phe Leu Gly Asn Glu Ser Pro 35 40 45 Thr Arg ArgArg Tyr Cys Gly Gly Lys Ala Gly Thr Phe Val Lys Ala 50 55 60 Ile Gly ArgLys Glu Gly Lys Ser Met Gly Ser Arg Ser Ser Ser Leu 65 70 75 80 Asp AlaAsp Asp Ser Gly Leu Gly Glu Glu Ala Ser Leu Ser Asp Asn 85 90 95 Ser AspLeu Asn Glu Asn Glu Pro Lys Lys His Val Asn Arg His Lys 100 105 110 IleLys Val Thr Thr Met Gly Asp Leu Arg Ser Arg Trp Gln Arg Phe 115 120 125Ala Glu Asp His Met Glu Gly Gln Lys Leu Asn Pro Phe Ser Glu Glu 130 135140 Phe Asp Tyr Asp His Ala Met Ala Thr Arg Leu His Lys Gly Asp Ala 145150 155 160 Gly Tyr Gly Arg Pro Lys Lys Asp Pro Lys Gln Leu Ser Glu GlnIle 165 170 175 Glu Pro Lys Ser Thr Ser Thr Ala Arg Trp Arg Arg Cys AlaSer Ser 180 185 190 Tyr Glu Thr Trp Ala Ser Arg Thr Asn Arg Ala 195 2007 489 DNA Caenorhabditis elegans CDS (1)..(489) 7 atg tca att gca tgtgct aga att gat aaa aca att ttc aaa ttc aaa 48 Met Ser Ile Ala Cys AlaArg Ile Asp Lys Thr Ile Phe Lys Phe Lys 1 5 10 15 gaa atg gag cag aatgta gcg act cag agc aaa gat gat gtg tat tcc 96 Glu Met Glu Gln Asn ValAla Thr Gln Ser Lys Asp Asp Val Tyr Ser 20 25 30 aaa gat ttt act caa aagaaa atg gac aag tcc agt agc gaa tat gga 144 Lys Asp Phe Thr Gln Lys LysMet Asp Lys Ser Ser Ser Glu Tyr Gly 35 40 45 cgg cca aaa cca gga act cttaca gag caa aga gct aaa aaa gct gcc 192 Arg Pro Lys Pro Gly Thr Leu ThrGlu Gln Arg Ala Lys Lys Ala Ala 50 55 60 gcc cac gtt cac aga gaa atg ctaaca tta tgt gaa gtt gtg gag gat 240 Ala His Val His Arg Glu Met Leu ThrLeu Cys Glu Val Val Glu Asp 65 70 75 80 tat ggt aaa caa gag aag gaa ggagat cca atc aga atc aca ttt gga 288 Tyr Gly Lys Gln Glu Lys Glu Gly AspPro Ile Arg Ile Thr Phe Gly 85 90 95 aga ctt ttc aca att tat gtc aat atttct gat aag gta gtt gga acc 336 Arg Leu Phe Thr Ile Tyr Val Asn Ile SerAsp Lys Val Val Gly Thr 100 105 110 ctt ttg cga gct cgt aaa cac aaa atgata gat ttt gaa gga gaa atg 384 Leu Leu Arg Ala Arg Lys His Lys Met IleAsp Phe Glu Gly Glu Met 115 120 125 tta ttt caa aag aga gat gat cat gttatt atc aca ctt tta ctc tct 432 Leu Phe Gln Lys Arg Asp Asp His Val IleIle Thr Leu Leu Leu Ser 130 135 140 gga gca cag ctt aaa gag gct att cgagca cac gca gca gca aac cca 480 Gly Ala Gln Leu Lys Glu Ala Ile Arg AlaHis Ala Ala Ala Asn Pro 145 150 155 160 aag gaa taa 489 Lys Glu 8 162PRT Caenorhabditis elegans 8 Met Ser Ile Ala Cys Ala Arg Ile Asp Lys ThrIle Phe Lys Phe Lys 1 5 10 15 Glu Met Glu Gln Asn Val Ala Thr Gln SerLys Asp Asp Val Tyr Ser 20 25 30 Lys Asp Phe Thr Gln Lys Lys Met Asp LysSer Ser Ser Glu Tyr Gly 35 40 45 Arg Pro Lys Pro Gly Thr Leu Thr Glu GlnArg Ala Lys Lys Ala Ala 50 55 60 Ala His Val His Arg Glu Met Leu Thr LeuCys Glu Val Val Glu Asp 65 70 75 80 Tyr Gly Lys Gln Glu Lys Glu Gly AspPro Ile Arg Ile Thr Phe Gly 85 90 95 Arg Leu Phe Thr Ile Tyr Val Asn IleSer Asp Lys Val Val Gly Thr 100 105 110 Leu Leu Arg Ala Arg Lys His LysMet Ile Asp Phe Glu Gly Glu Met 115 120 125 Leu Phe Gln Lys Arg Asp AspHis Val Ile Ile Thr Leu Leu Leu Ser 130 135 140 Gly Ala Gln Leu Lys GluAla Ile Arg Ala His Ala Ala Ala Asn Pro 145 150 155 160 Lys Glu 9 399PRT Drosophila melanogaster 9 Met Thr Asp Val Ser His Glu Leu Gly AlaLeu Arg Phe Val Val Leu 1 5 10 15 Arg Tyr Leu Gln Asp Ser Pro Leu SerSer Lys Val Ala Met Phe Asn 20 25 30 Asn Gln Ala Thr Gln His Lys Gln SerGln Leu Leu Asn Pro Phe Ser 35 40 45 Gln Asp Gly Arg Ala Ala Ser Pro LysPro Thr Phe Ser Lys Asp Gln 50 55 60 Tyr Gly Lys Pro Leu Ala Gly Ser LeuThr Glu Met Arg Gly Gln Lys 65 70 75 80 Ala Asn Ile His Val Met Lys GluMet Leu Glu Leu Cys Gln Ile Ile 85 90 95 Asn Ser Glu Gly Tyr Asp Val LysAsp Glu Pro Thr Met Arg Val Ile 100 105 110 Pro Phe Gly Glu Leu Phe AsnVal Ser Val Leu Phe Thr Ala Gly Ile 115 120 125 Phe Phe Glu Lys Pro SerLys Leu Val Thr Ser Thr Leu Gln Ile Tyr 130 135 140 Asn Tyr Ile Ser AspLys Val Val Gly Ile Leu Leu Arg Ala Arg Lys 145 150 155 160 His Lys LeuVal Asp Phe Glu Gly Glu Met Leu Tyr Gln Arg Arg Asp 165 170 175 Asp AspVal Pro Val Phe Leu Leu Lys Pro Ile Lys Glu Ile Arg Ser 180 185 190 GluMet Glu Ala Lys Ile Glu Asp Ile Lys Arg Ala Ala Ser Pro Ala 195 200 205Pro Pro Gln Ser Thr Ser Val Leu Met Asp Arg Ser Ala His Glu Gln 210 215220 Lys Leu Lys Ser Arg Thr Pro Ser Pro Ala Val Gly Lys Ser Ala Lys 225230 235 240 Ser Lys Ser Ala Ser Pro Ala Pro Lys Ala Pro Val Pro Val ProAla 245 250 255 Pro Ala Ala Glu Val Thr Pro Val Ala Gly Pro Thr Thr SerAla Glu 260 265 270 Pro Ala Pro Val Ala Glu Ser Thr Met Ala Ala Val ProAla Pro Ser 275 280 285 Thr Glu Pro Thr Pro Ala Thr Ala Pro Ala Ser SerThr Val Glu Ile 290 295 300 Glu Pro Ala Lys Pro Glu Val Thr Glu Gln AlaPro Val Ala Val Ile 305 310 315 320 Val Thr Glu Ala Pro Ser Thr Glu GluThr Thr Pro Thr Thr Ser Glu 325 330 335 Pro Gln Ala Glu Glu Ala Pro AlaAla Val Ala Pro Ala Gly Pro Ala 340 345 350 Asp Asp Leu Pro Thr Ile ValIle Glu Ala Thr Ala Glu Phe Val Arg 355 360 365 Thr Val Ser Val Glu GlnLeu Ala Pro Ser Pro Gly Thr Ala Ser Glu 370 375 380 Ser Ser Pro Asp GlnSer Gln Ser Gln Pro Glu Ser Thr Pro Ala 385 390 395 10 12 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 10gatctgcggt ga 12 11 24 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 11 agcactctcc agcctctcac cgca 24 12 12 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer12 gatctgttca tg 12 13 24 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 13 accgacgtcg actatccatg aaca 24 1413 DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 14 gatctttcca tcg 13 15 24 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 15 aggcaactgt gctatccgag ggaa 24

What is claimed is:
 1. An isolated polynucleotide encoding a STARSpolypeptide.
 2. The isolated polynucleotide of claim 1, wherein theSTARS polypeptide comprises an amino acid sequence of SEQ ID NO:2, 4, 6,8 or
 10. 3. The polynucleotide of claim 1, wherein said polynucleotidehas a nucleic acid sequence of SEQ ID NO:1, 3, 5, 7 or 9, or acomplement thereof.
 4. The polynucleotide of claim 2, wherein saidpolynucleotide further comprises a promoter operable in eukaryoticcells.
 5. The polynucleotide of claim 4, wherein said promoter isselected from the group consisting of hsp68, SV40, CMV, MKC, GAL4_(UAS),HSV and β-actin.
 6. The polynucleotide of claim 5, wherein said promoteris a tissue specific promoter.
 7. A nucleic acid of 15 to about 2000base pairs comprising at least 15 contiguous base pairs of SEQ ID NO:1,3, 5, 7 or 9, or the complement thereof.
 8. The nucleic acid of claim 7,comprising 20 contiguous base pairs of SEQ ID NO:1, 3, 5, 7 or 9, or thecomplement thereof.
 9. The nucleic acid of claim 7, comprising 25contiguous base pairs of SEQ ID NO:1, 3, 5, 7 or 9, or the complementthereof.
 10. The nucleic acid of claim 7, comprising 30 contiguous basepairs of SEQ ID NO:1, 3, 5, 7 or 9, or the complement thereof.
 11. Thenucleic acid of claim 7, comprising 50 contiguous base pairs of SEQ IDNO:1, 3, 5, 7 or 9, or the complement thereof.
 12. The nucleic acid ofclaim 7, comprising 100 contiguous base pairs of SEQ ID NO:1, 3, 5, 7 or9 or the complement thereof.
 13. The nucleic acid of claim 7, comprising150 contiguous base pairs of SEQ ID NO:1, 3, 5, 7 or 9, or thecomplement thereof.
 14. The nucleic acid of claim 7, comprising 250contiguous base pairs of SEQ ID NO:1, 3, 5, 7 or 9, or the complementthereof.
 15. The nucleic acid of claim 7, comprising 500 contiguous basepairs of SEQ ID NO:1, 3, 5, 7 or 9, or the complement thereof.
 16. Thenucleic acid of claim 7, comprising 1000 contiguous base pairs of SEQ IDNO:1, 3, 5, 7 or 9, or the complement thereof.
 17. The nucleic acid ofclaim 7, comprising 2000 contiguous base pairs of SEQ ID NO:1, 2, 5, 7or 9, or the complement thereof.
 18. A peptide comprising 10 contiguousamino acids of SEQ ID NO:2, 4, 6, 8 or
 10. 19. The peptide of claim 18,comprising 15 contiguous amino acids of SEQ ID NO:2, 4, 6, 8 or
 10. 20.The peptide of claim 18, comprising 20 contiguous amino acids of SEQ IDNO:2, 4, 6, 8 or
 10. 21. The peptide of claim 18, comprising 25contiguous amino acids of SEQ ID NO:2, 4, 6, 8 or
 10. 22. The peptide ofclaim 18, comprising 30 contiguous amino acids of SEQ ID NO:2, 4, 6, 8or
 10. 23. The peptide of claim 18, comprising 50 contiguous amino acidsof SEQ ID NO:2, 4, 6 or
 8. 24. An expression construct comprising apolynucleotide encoding a STARS polypeptide operably linked to aregulatory sequence.
 25. The expression construct of claim 24, whereinthe polynucleotide encodes a STARS polypeptide comprising an amino acidsequence of SEQ ID NO:2, 4, 6, 8 or
 10. 26. The expression construct ofclaim 25, wherein said regulatory sequence is a tissue specificpromoter.
 27. The expression construct of claim 26, wherein saidpromoter is a muscle specific promoter.
 28. The expression construct ofclaim 27, wherein said muscle specific promoter is selected from thegroup consisting of myosin light chain-2 promoter, α actin promoter,troponin 1 promoter, Na⁺/Ca²⁺ exchanger promoter, dystrophin promoter,creatine kinase promoter, α7 integrin promoter, brain natriureticpeptide promoter, αB-crystallin/small heat shock protein promoter, αmyosin heavy chain promoter and atrial natriuretic factor promoter. 29.The expression construct of claim 25, wherein said promoter is aninducible promoter.
 30. The expression construct of claim 25, whereinsaid expression construct is contained in a viral vector.
 31. Theexpression construct of claim 25, wherein said viral vector is selectedfrom the group consisting of a retroviral vector, an adenoviral vector,and adeno-associated viral vector, a vaccinia viral vector, aherpesviral vector, a polyoma viral construct or a Sindbis viral vector.32. The expression construct of claim 24, wherein said expressionconstruct comprises a polyadenylation signal.
 33. The expressionconstruct of claim 24, wherein said expression construct comprises asecond polynucleotide encoding a second polypeptide.
 34. The expressionconstruct of claim 32, wherein said second polynucleotide is under thecontrol of a second regulatory sequence.
 35. A polypeptide comprisingthe sequence of SEQ ID NO:2, 4, 6, 8 or
 10. 36. A method of screeningfor modulators of STARS expression comprising: (a) providing a cell inwhich a STARS promoter directs the expression of a polypeptide; (b)contacting said cell with a candidate modulator; and (c) measuring theeffect of said candidate modulator on said polypeptide, wherein adifference in expression of said polypeptide, as compared to anuntreated cell, indicates that said candidate modulator is a modulatorof STARS expression.
 37. The method of claim 36, wherein measuringcomprises Northern analysis.
 38. The method of claim 36, whereinmeasuring comprise PCR.
 39. The method of claim 36, wherein measuringcomprises RT-PCR.
 40. The method of claim 36, wherein measuringcomprises immunologic detection of STARS.
 41. The method of claim 36,wherein measuring comprises ELISA.
 42. The method of claim 36, whereinmeasuring comprises immunohistochemistry.
 43. The method of claim 36,wherein said cell is located in an animal.
 44. The method of claim 36,wherein said cell is a myocyte.
 45. The method of claim 44, wherein saidcell is a cardiomyocyte.
 46. The method of claim 36, wherein saidmodulator decreases expression of the polypeptide.
 47. The method ofclaim 36, wherein said modulator increases expression of thepolypeptide.
 48. The method of claim 36, wherein said polypeptide isSTARS.
 49. The method of claim 36, wherein said polypeptide is ascreenable marker polypeptide.
 50. The method of claim 49, wherein saidscreenable marker polypeptide is luciferase, β-galactosidase, CAT orgreen fluorescent protein.
 51. A method of screening for modulators ofSTARS actin-binding activity comprising: (a) providing an active STARSpreparation; (b) contacting said STARS preparation with a candidatemodulator; and (c) measuring the actin binding activity of said STARSpreparation, wherein a difference in actin binding activity of saidSTARS preparation, as compared to an untreated STARS preparation,indicates that said candidate modulator is a modulator of STARS actinbinding activity.
 52. The method of claim 51, wherein said method isperformed in a cell free assay.
 53. The method of claim 51, wherein saidmethod is performed in a cell.
 54. The method of claim 51, whereinbinding is determined by chromatographic separation.
 55. The method ofclaim 51, wherein binding is determined by electrophoretic separation.56. A method of screening for an inhibitor of STARS-inducedtranscription comprising: (a) providing a cell that expresses STARS andcontains a STARS-regulated promoter linked to an indicator gene; (b)contacting said cell with a candidate modulator; and (c) measuring theeffect of said candidate modulator on expression of said indicator gene,wherein a difference in expression of said indicator gene, as comparedto an untreated cell, indicates that said candidate modulator is amodulator of STARS-induced transcription.
 57. The method of claim 56,wherein said cell is a myocyte.
 58. The method of claim 56, wherein saidcell is a cardiomyocyte.
 59. The method of claim 56, wherein saidSTARS-regulated promoter is SM22.
 60. The method of claim 56, whereinsaid indicator gene encodes luciferase, β-galactosidase, CAT or greenfluorescent protein.
 61. A method of producing a STARS polypeptide in acell comprising: (a) transforming a cell with an expression cassettecomprising a nucleic acid encoding STARS under the control of a promoteractive in said cell; (b) culturing said cell under conditions suitablefor expression of STARS.
 62. The method of claim 61, wherein said cellis a cardiomyocyte or a fibroblast, such as a cardiac fibroblast. 63.The method of claim 61, wherein said cell is located in an animal. 64.The method of claim 61, wherein transforming comprises infection with aviral vector.
 65. The method of claim 61, wherein transforming comprisescontacting of said cell with a liposome comprising said expressioncassette.
 66. The method of claim 61, wherein transforming compriseselectroporation, calcium phosphate precipitation or protoplast fusion.67. The method of claim 61, wherein said cell is a prokaryotic cell. 68.The method of claim 61, wherein said cell is a eukaryotic cell.
 69. Themethod of claim 61, further comprising the step of purifying said STARSpolypeptide away from other cellular components.
 70. A non-humantransgenic animal comprising a selectable or screenable marker proteinunder the control of a STARS promoter.
 71. A non-human transgenic animalcomprising a STARS encoding nucleic acid under the control of aninducible promoter.
 72. A non-human transgenic animal comprising a STARSencoding nucleic acid under the control of a constitutive promoter. 73.A non-human transgenic animal lacking at least one STARS allele.
 74. Thenon-human transgenic animal of claim 73, wherein said animal lacks bothalleles of STARS.
 75. A method of inhibiting STARS activity comprisingcontacting a cell expressing STARS with a compound that inhibits STARSactivity.
 76. The method of claim 75, wherein said compound is a nucleicacid antisense to a STARS regulatory or coding region.
 77. The method ofclaim 75, wherein said compound is a ribozyme that selectively cleaves aSTARS transcript.
 78. The method of claim 75, wherein said compound is asmall molecule inhibitor.
 79. The method of claim 75, wherein saidcompound is a single chain antibody that binds immunologically to STARS.80. A method of treating cardiac hypertrophy and dilated cardiomyopathycomprising decreasing STARS activity in heart cells of a subject. 81.The method of claim 80, wherein STARS activity is decreased bydelivering an expression vector comprising a polynucleotide encoding anantisense STARS construct, a STARS ribozyme or an anti-STARSsingle-chain antibody to said subject.
 82. The method of claim 81,wherein the expression vector is a non-viral vector.
 83. The method ofclaim 81, wherein the expression vector comprises a viral vector. 84.The method of claim 83, wherein said viral vector is an adenoviralconstruct, a retroviral construct, an adeno-associated viral construct,a herpesviral construct, a vaccinia viral construct, a polyoma viralconstruct or a Sindbis viral vector.
 85. The method of claim 84, whereinthe viral vector comprises a replication-defective adenovirus.
 86. Themethod of claim 81, wherein the step of delivering the expressionconstruct comprises introducing a viral vector comprising the nucleicacid into the heart of the mammal by direct injection into the hearttissue.
 87. The method of claim 81, wherein the step of delivering theexpression construct comprises introducing the expression construct intothe lumen of at least one vessel supplying blood to the heart.
 88. Themethod of claim 81, further comprising administering a secondanti-hypertophic drug to said subject.
 89. A method of treatingmyocardial infarct comprising decreasing STARS activity in heart cellsof a subject.
 90. A method of preventing cardiac hypertrophy and dilatedcardiomyopathy comprising decreasing STARS activity in heart cells of asubject.
 91. A method of inhibiting progression of cardiac hypertrophycomprising decreasing STARS activity in heart cells of a subject.
 92. Amethod of treating heart failure comprising decreasing STARS activity inheart cells of a subject.
 93. A method of inhibiting progression ofheart failure comprising decreasing STARS activity in heart cells of asubject.
 94. A method of increasing exercise tolerance in a subject withheart failure or cardiac hypertrophy comprising decreasing STARSactivity in heart cells of a subject.
 95. A method of reducinghospitalization in a subject with heart failure or cardiac hypertrophycomprising decreasing STARS activity in heart cells of a subject.
 96. Amethod of improving quality of life in a subject with heart failure orcardiac hypertrophy comprising decreasing STARS activity in heart cellsof a subject.
 97. A method of decreasing morbidity in a subject withheart failure or cardiac hypertrophy comprising decreasing STARSactivity in heart cells of a subject.
 98. A method of decreasingmortality in a subject with heart failure or cardiac hypertrophycomprising decreasing STARS in heart cells of a subject.
 99. A method ofproducing a modulator of STARS expression comprising: (a) providing acell in which a STARS promoter directs the expression of a polypeptide;(b) contacting said cell with a candidate modulator; (c) measuring theeffect of said candidate modulator on said polypeptide, wherein adifference in expression of said polypeptide, as compared to anuntreated cell, indicates that said candidate modulator is a modulatorof STARS expression; and (d) producing said modulator.
 100. A method ofproducing a modulator of STARS actin binding activity comprising: (a)providing an active STARS preparation; (b) contacting said STARSpreparation with a candidate modulator; (c) measuring the actin bindingactivity of said STARS preparation, wherein a difference in actinbinding activity of said STARS preparation, as compared to an untreatedSTARS preparation, indicates that said candidate modulator is amodulator of STARS acting binding activity; and (d) producing saidmodulator.
 101. A modulator of STARS expression identified according tothe method comprising: (a) providing a cell in which a STARS promoterdirects the expression of a polypeptide; (b) contacting said cell with acandidate modulator; and (c) measuring the effect of said candidatemodulator on said polypeptide, wherein a difference in expression ofsaid polypeptide, as compared to an untreated cell, indicates that saidcandidate modulator is a modulator of STARS expression.
 102. A modulatorof STARS actin binding activity identified according to the methodcomprising: (a) providing a STARS preparation; (b) contacting said STARSpreparation with a candidate modulator; and (c) measuring the actinbinding activity of said STARS preparation, wherein a difference inactin binding activity of said STARS preparation, as compared to anuntreated STARS preparation, indicates that said candidate modulator isa modulator of STARS actin binding activity.
 103. An antibody that bindsimmunologically to STARS.
 104. A polyclonal antibody preparation,antibodies of which bind immunologically to STARS.
 105. A hybridoma cellthat produces a monoclonal antibody that binds immunologically to STARS.